Abstract:
An optical spectrum analyzer ( 100 ) having a an excitation light source ( 103 ) with a luminated bio-sample ( 114 ) carried by a flow path ( 102 ). A spectrum dispersive element  213  dispersants lumineses light generated by the bio-sample ( 114 ).

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a nonprovisional of U.S. Provisional Patent Application No. 62/089864 filed on Dec. 10, 2014. This application incorporates the disclosure of such application in its entirety by reference. To the extent that the present disclosure conflicts with the referenced application, however, the present disclosure is to be given priority. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention generally relates to cytometers; and more particularly, flow-cytometers and methods of making, design, use, and integrating, these cytometers into systems. 
       BACKGROUND 
       [0003]    Generally, flow-cytometry is a method for counting, examining, sorting, measurement of and characterization of various aspects of microscopic particles, bio-particles, bio-cells, and their derivatives to determine their physical and/or chemical characteristic via an optical and/or electronic detection apparatus. The early development of a flow-cytometric system was based on impedance and was developed by Wallace H. Coulter in 1953. Over the years, several developments where made by Mack Fulwyler, Wolfgang Gohde, and others that increased the system&#39;s useful acceptability into the marketplace. In today&#39;s conventional flow-cytometry, the flow-cytometery is generally based on the use of light in the visible spectrum.  
         [0004]    Typicality, a flow-cytometer is an analytical instrument that emits a certain frequency or frequencies of light that are directed toward a sample or samples. The light emitted from the light source excites the sample to emit a certain frequency or frequencies of light from the surface of the sample of the sample and in some cases from inside the sample. The frequencies of light that are emitted form the surface of the sample and in some cases emitted from the interior of the sample are collected and analyzed. 
         [0005]    However, certain problems such as non-integration of certain parts into unitary whole or unitary design has made the adoption of conventional flow-cytometric machines slow to come into the marketplace. Also, because of this poor design, conventional flow-cytometers are not capable of accurately counting and characterizing cells, bio-cells cells, and/or biologic materials, thereby limiting the use and potential usefulness of conventional flow-cytometric instruments. Moreover, since this poor design causes other problems such as manufacturing, reliability, and the like which degrades the usefulness of conventional flow-cytometers in the marketplace. Further, the poor design of conventional flow-cytometers limits the manufacturing capability and cost parameters which drives up costs. Thus, limiting the manufacturing capability and making conventional flow-cytometers more expensive. 
         [0006]    It can be readily seen that conventional flow-cytometers have several problems and disadvantages. Despite many potential advantages of flow-cytometery, market acceptance is limited, especially in portable and non-portable applications. Further, since some of the applications of flow-cytometry are high volume applications, theses problems and disadvantages do not allow conventional flow-cytometric technology to be used so as to drive the cost of flow-cytometry lower and to be more useful in high volume applications. Therefore, a low cost flow-cytometric system or instrument capable with high volume manufacturability and better efficiency would be highly desirable. 
       SUMMARY OF THE INVENTION 
       [0007]    In various representative aspects, the present invention provides an optical spectrum analyzer having an excitation light source that illuminates a bio-sample. The bio-sample luminescence&#39;s with the excitation of the bio-sample by the excitation light source. The luminescence is directed to a dispersal element wherein the luminescence is spread and digitized, and stored and analyzed in a digital signal processing unit. The digital signal process unit is directly and intimately in communication with the initial acquisition of the of the luminescence data which allows the flow-cytometer to be used as a counter. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0008]    Representative elements, operational features, applications and/or advantages of the present invention reside inter alia in the details of construction and operation as more fully hereafter depicted, described and claimed—reference being made to the accompanying drawings forming a part hereof, wherein like numerals refer to like parts throughout. Other elements, operational features, applications and/or advantages will become apparent to skilled artisans in light of certain exemplary embodiments recited in the Detailed Description, wherein: 
           [0009]      FIG. 1  is a schematic illustration of a conventional simplified flow-cytometer showing an excitation light source, a flow path, a plurality of multiple photo-detectors, multiple filters, multiple gates, and a digital signal processor, and a triggering device; 
           [0010]      FIG. 2  is a simplified schematic illustration of an embodiment of a flow-cytometer illustrating the present invention and showing an excitation light source a flow path, a plurality of multiple photo-detectors, and a digital signal processor, and a triggering device; and 
           [0011]      FIG. 3  is a simplified schematic illustration of another embodiment of the present invention illustrating a simplified flow-cytometer showing an excitation light source, a flow path, a plurality of multiple photo-detectors, and a digital signal processor, and a triggering device. 
       
    
    
       [0012]    Those skilled in the art will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures may be exaggerated relative to other elements to help improve understanding of various embodiments of the present invention. Furthermore, the terms ‘first’, ‘second’, and the like herein, if any, are used inter alia for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. Moreover, the terms front, back, top, bottom, over, under, and the like in the Description and/or in the claims, if any, are generally employed for descriptive purposes and not necessarily for comprehensively describing exclusive relative position. Skilled artisans will therefore understand that any of the preceding terms so used may be interchanged under appropriate circumstances such that various embodiments of the invention described herein, for example, are capable of operation in other orientations than those explicitly illustrated or otherwise described. 
       DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       [0013]    Before addressing details of embodiments described below, some terms are defined or clarified. 
         [0014]    The term “filter” is intended to mean any material or device that is capable of filtering, impeding, or preventing certain frequencies of light to pass thought the filter. 
         [0015]    The term “emission detector” is intended to any device or material that is capable of detecting and/or sensing emission from the sample material. Typically, an emission detector can sense or detect a variety of frequencies. However, it should be understood that the emission detector can made to sense or detect on a single frequency of light or a small group of frequencies of light. Additionally, the emission detector and be made with a variety of emission detectors. 
         [0016]    The term “sample” is intended to mean any material or materials, any biologic or non-biologic material that is capable of emitting light or being luminescent from the sample being exited from the source. 
         [0017]    The term “bio-sample” is intended to mean any biologic material or materials, is capable of emitting light or luminesing from the sample being exited from the source. 
         [0018]    The term “bio-cell(s)” is intended to mean a fundamental biologic unit that can be alive and/or dead. 
         [0019]    The term “cell(s)” is intended to mean a fundamental unit that can either be alive of not alive. By way of example only, but not limited to, a virus, a mycoplasem, or the like are sometimes hard to define. 
         [0020]    The term “luminescence spectrum” is intended to mean a spectrum of bright lines, bands, or continuous radiation characteristics of and determined by a specific emitting substances subjected to a specific kind of excitation. 
         [0021]    The term “luminescence signal capturing” is intended to mean the capture and recording of output signals from the illumination of the bio-sample by the light source which causes the bio-sample luminance at certain frequencies that allow certain characteristics to be identified and recorded. 
         [0022]    The term “excitation light source” is intended to mean any suitable light source that is capable of illuminating a sample and emitting luminescence. 
         [0023]    The term “flow path” is intended to mean any suitable liquid that is capable of suspending and carrying a sample to a region wherein the sample can be illuminated by the excitation light source. 
         [0024]    The term “digital signal processor” (DSP) (also known as a Digital signal device) is intended to mean a specialized microprocessor (or a SIP block), with its architecture optimized for the operational needs of digital signal processing. Typically, the goal of DSPs is usually to measure, filter and/or compress continuous real-world analog signals. 
         [0025]    As used herein, the terms “comprises”, “comprising”, or any variation thereof, are intended to reference a non-exclusive inclusion, such that a process, method, article, composition or apparatus that comprises a list of elements does not include only those elements recited, but may also include other elements not expressly listed or inherent to such process, method, article, composition or apparatus. Other combinations and/or modifications of the above-described structures, arrangements, applications, proportions, elements, materials or components used in the practice of the present invention, in addition to those not specifically recited, may be varied or otherwise particularly adapted by those skilled in the art to specific environments, manufacturing specifications, design parameters or other operating requirements without departing from the general principles of the same. 
         [0026]    Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present and B is true (or present, and both A and B are true (or present) Also, use of the “a” or “an” are employed to describe elements and components of the invention. This is done merely for convenience and to give a general sense of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise. 
         [0027]    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
         [0028]    To the extent not described herein, many details regarding specific materials, processing acts, and circuits are conventional and may be found in textbooks and other sources within the fuel cell and chemical arts. 
         [0029]    Referring now to  FIG. 1 ,  FIG. 1  is conventional flow-cytometer  100  incorporating an optical spectrum analyzer to analyze bio molecules that are excited to luminescence, indicted by arrows  109  and  111 . Such luminescence&#39;s can be generated either by auto-luminescent from the cell itself or from a color label tag, or marker, attached to the bio cell or cells. Information about the bio cell or cells can be obtained though the analysis of spectrum, which includes its wavelength distribution and intensity different spectrum distribution indicate the presence of different bio-cells and/or markers on the bio-cell(s). Additionally, its corresponding intensity indicates the concentration of such distinct bio cells or distinct marker on the bio-cell(s). 
         [0030]    Typical bio-samples  114  may include varieties of bio-cell molecules, each corresponding to a unique spectral distribution through either auto-luminescence generation or a labeled color tag. Conventional flow-cytometer  100  incorporates multiple photo-detectors  128 ,  130 ,  132 ,  134 , and  126 , such as photomultiplier tubes (PMTS), with filters  138 ,  140 ,  142 , and  144  which can be customized to receive the correspondent luminescence spectrum  109  and a plurality of spectrum  111  and their intensity and reject the rest, as shown in  FIG. 1 . One photo-detector such as photo-detector  132  thus corresponds to one type of bio-cell molecule or marker on the bio-cell. Conventional flow-cytometer  100  includes a liquid flow path  102  containing bio-cell samples  114 . Excitation light source  103 , such as a semiconductor laser or LED, is used to excite bio samples  114  in region  107  on flow path  102 . The bio-cells at region  107  emit luminescence  111 , which is collected by multiple photo-detectors  131 - 134  and  126  with photo-detectors  128 ,  130 ,  132 , and  134  having correspondent filters  138 ,  140 ,  142 , and  144  to allow only interested luminescence spectral contents to be detected by any individual photo-detector within  128 ,  130 ,  132 , and  134 . The output analog signals of the photo-detector  128 ,  130 ,  132 , and  134  are digitized by the digital signal processing (DSP)  141  circuitry, which includes multiple A/D converters. Scattering light  109  from the bio-sample region  107  is captured by a photo-detector  126 , which generates triggering signal  143  to trigger DSP  141  to synchronize the luminescence signal capturing. This also allows counting of the bio cells or markers on the bio cells in addition to recording their emitted luminescence under light excitation. Additionally, crosstalk between different markers is compensated by complicated calibration procedures. 
         [0031]    Different bio-cell molecules emit different characteristic luminescence due to its own unique auto-luminescence or the attached color labels. The luminescence spectrums may overlap with each other if they are too close to each other in wavelengths. This crosstalk limits the detection sensitivity and the simultaneous detection of multiple types of bio cells or markers on the bio cells. 
         [0032]    Referring now to  FIG. 2 ,  FIG. 2  is a simplified schematic illustration of a flow-cytometer  200  that has been simplified so as to better illustrate the present invention.  FIG. 2  shows an excitation light source  203 , a flow path  201 , a plurality photo-detectors  215 , with individual photo-detectors  205 ,  221 ,  222 ,  223 ,  224 ,  225 ,  226 ,  227 ,  228 , and  229  individually identified, a digital signal processor  241 , and a triggering device  243 . Further, as shown in  FIG. 2 , flow-cytometer  200  includes a liquid flow path  201  having a plurality of bio-samples  214 , illustrated by individual bio-samples  216 ,  218 , and  220 . The plurality of bio-samples  214  are typically suspended in any suitable liquid, suspension, or liquid medium capable of carrying the plurality of bio-samples  214  to region  207 . 
         [0033]    Generally, any suitable liquid suspension or liquid medium can be used such as, but not limited to, saline, five percent dextrose, buffering solution, or the like. As illustrated in  FIG. 2 , flow path  201  is indicated by a plurality of flow lines  204 , identified individually and in part by flow lines  106 ,  108 , and  110 . 
         [0034]    As shown in  FIG. 2 , excitation light source  203  is used to illuminate region  207  wherein individual bio samples  114  are illuminated as the bio-samples pass though region  207 . Excitation light source  203  can be made of any suitable device such as, a light emitting diode, light emitting photo-diode, a laser, a semiconductor laser, or the like. Moreover, excitation light source  203  can be configured to produce any suitable wavelength or wavelengths of light that is desired. By way of example, after the plurality of bio-samples  215  are illumined by excitation light source  203 , the plurality of bio-samples luminescence (emit light at a different wavelength, generally a lower wavelength). 
         [0035]    Typically, the emitted light from the luminescence of the bio-sample is collected by any suitable photo-detector or group of photo-detectors. As shown in  FIG. 2 , individual photo-detectors  205 ,  221 ,  223 ,  225 ,  227 ,  228 ,  230 ,  231  and  232  can be configured so as to capture light individually or can be configure so as to capture light as a plurality of photo-detectors  215  depending upon the specific application and/or need. 
         [0036]    Any suitable photo-detector can be used such as, but not limited to, a semiconductor photo-detectors, Active-pixel sensors (APSs), Charged-coupled devices (CCD), or the like. 
         [0037]    As shown in  FIG. 2  an array of photo-detectors  215  can be used to record full bio-molecule spectrum  211  dispersed spatially by a dispersive element  213 , such as a grating or a prism, as shown in  FIG. 2 . Luminescence spectrum of a color label or auto luminescence typically follows its own characteristic profile that can be pre-calibrated. When a bio-sample including multiple types of bio-cells is excited at region  207  by excitation light source  203 , the characteristic luminescence profiles will overlap, and the plurality of photo-detectors  215  or photo-detector array  215  will record the combined luminescence spectrums  211  through the individual photo-detectors  221 ,  222 ,  223 ,  224 ,  225 ,  226 ,  227 ,  228 , and  229 . Additionally, it should be understood that any suitable sized array could be used depending upon the specific application. Each photo-detector component receives a particular spectrum content of the spectrum distribution. As the characteristic luminescence spectrum of any particular color label and auto-luminescence has already been pre-calibrated, the distribution intensity for each bio cell can be de-convoluted from the combined spectrums through digital data processing after the received analog signals are converted into digital data by the DSP  241 . This will allow the use of more color labels closely spaced between each other in peak wavelength, thus increasing the types of bio-cells to be detected simultaneously without sacrificing the detection sensitivity. In this detection method, conventional hardware filters are not needed as the full spectrum intensity will be extrapolated and thus, a software filter can be implemented in data processing to obtain any data equivalent to that from a hardware filter in a conventional flow-cytometer. Software implementation is flexible and can be automated, thus simplifying the overall system and reducing both the manufacturing and the service cost. 
         [0038]    In the array detection scheme, the data converted by DSP  241  is synchronized by the conventional way, i.e. by scattered excitation light or luminescence  209 , which is received by a separate photo-detector  205  to generate trigger signal  243 . Bio samples  214  are usually complicated and include varieties of bio cells and particles, which will all scatter light. Some of those events are of interest and others are not. When using scattered light as a trigger for the array signal capture synchronization, all of those events are counted which increases the data storage space size and slows down the data analysis. When two events of interest are spaced too close in time during the analysis, those events will be discarded because the system cannot distinguish them as two separate valid events. Alternatively, luminescence signal from a separated photo-detector has been used as the trigger, and only events of interest are captured, thus improving the data analysis throughput. However, there are two deficiencies in this approach: 
         [0039]    a When two events of interest are spaced too close to each other in timing, the system will miss one valid event. 
         [0040]    b Extra photo detector for luminescence detection increases the system manufacturing cost. 
         [0041]    It is the purpose of this invention to correct the deficiencies of existing flow cytometer by using received florescence light from the array photo-detector to trigger the data capture and analysis. This self-triggering is possible because array photo-detector is designed to receive full luminescence signals without any optical filtering. When a valid bio cell or particle is excited by a light source, it will emit auto luminescence as the background in addition to the luminescence from the attached color label. Conventional flow cytometer removes certain unwanted luminescence using an optical filter, and thus, making self-triggering not practical. With the array photo-detector that captures complete optical spectrums, self-triggering becomes feasible. 
         [0042]    Referring now to  FIG. 3 ,  FIG. 3  shows a simplified schematic illustration of another embodiment of the present invention illustrating a simplified flow-cytometer  300  showing an excitation light source  303 , a flow path  301 , a plurality of multiple photo-detectors  315 , and a digital signal processor  341 , and a triggering device  343 . 
         [0043]    As shown in  FIG. 3 , flow-cytometer  300  shows an excitation light source  303  that excites bio-samples  314  in region  307  in the flow path  301 , which emit luminescence. A dispersive element  313 , such as grating or prism, spreads the luminescense spectrum  311  onto array photo-detector  315  with array components  321 ,  322 ,  323 ,  324 ,  325 ,  326 ,  327 ,  328 , and  329 . The array photo-detector  315  can be made of avalanche photodiode (APD) or the normal PMT. An add-logic in DSP  341  after the array photo-detector integrates all signals received from the array elements, and the output signal ΣDn is used as trigger  343  to synchronize the data capturing by DSP 341 . This output ΣDn from the add-logic should be in synch with the excited laser source whenever it encounters a bio cell of interest or marker on the bio cells in the flow path as the interested bio cell will emit either auto-luminescence or luminescence from the attached color label, thus enabling the cell counting of the events of interest while recording the associated luminescence spectrum for either in-situ or after-test data processing. More sophisticated triggering can be adopted through logic-add of signals from selected array receiver components, thus allowing to target selected cells for analysis, improving system efficiency and reduce detection error when analyzing such cells. For example, if only certain bio cells are of interest during analysis and the color labels for these cells are known, only signals from the correspondent photo receiver array components will be logic added as signal trigger during the operation. 
         [0044]    We have thus provided a simple and effective low cost self-triggered flow cytometer to capture complete spectrum information and improve the throughput of cell counting while also allowing the operation in presence of large unwanted molecules without bio-sample washing and lysing, thus simplifying the sample preparation process 
         [0045]    The following descriptions are of exemplary embodiments of the invention and the inventors&#39; conceptions of the best mode and are not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following Description is intended to provide convenient illustrations for implementing various embodiments of the invention. As will become apparent, changes may be made in the function and/or arrangement of any of the elements described in the disclosed exemplary embodiments without departing from the spirit and scope of the invention. 
         [0046]    In the foregoing specification, the invention has been described with reference to specific exemplary embodiments; however, it will be appreciated that various modifications and changes may be made without departing from the scope of the present invention as set forth in the claims below. The specification and figures are to be regarded in an illustrative manner, rather than a restrictive one and all such modifications are intended to be included within the scope of the present invention. Accordingly, the scope of the invention should be determined by the claims appended hereto and their legal equivalents rather than by merely the examples described above. For example, the steps recited in any method or process claims may be executed in any order and are not limited to the specific order presented in the claims. Additionally, the components and/or elements recited in any apparatus claims may be assembled or otherwise operationally configured in a variety of permutations to produce substantially the same result as the present invention and are accordingly not limited to the specific configuration recited in the claims. 
         [0047]    Benefits, other advantages and solutions to problems have been described above with regard to particular embodiments; however, any benefit, advantage, solution to problems or any element that may cause any particular benefit, advantage or solution to occur or to become more pronounced are not to be construed as critical, required or essential features or components of any or all the claims.