Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Electro-optical investigation, e.g. flow cytometers
  • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• • G01N15/149—

Definitions

• flow cytometry apparatus and methods to process information incident to particles or cells entrained in a sheath fluid stream allowing assessment, differentiation, assignment, and separation of such particles or cells even at high rates of speed.
• Flow cytometry is a field which has existed for many years. Basically, flow cytometer systems act to position small amounts of a substance within a sheath fluid. Through hydrodynamic focusing and laminar flow, the substance is split into individual particles, cells, or the like. In many applications, sheath fluid together with its entrained substance exits a nozzle in a jet and free falls or is channeled in an optically transparent pathway for analysis. The sheath fluid may form droplets encapsulating individual particles which are separated and collected based upon assignment of differentiated particle characteristics.
• Another significant problem associated with conventional analysis and compensation of variables in flow cytometry can be the preservation of original signal data from an occurrence incident to the fluid stream prior to subsequent processing steps. It may not have been possible to preserve or store original signal data until now due to the short amount of time in which to analyze or compensate the original signal. As such, all or part of the original or raw signal data may have been sacrificed to increase the efficiency of analysis or provide feed back compensation events. The practice of discarding original raw data may prevent re- analysis of the data to improve quality control, to establish good manufacturing practices, and attain procedural thresholds for certain regulatory or statutory requirements.
• Yet another problem with conventional analysis may be the inability to process high speed serial occurrences, to compensate multiple parameters, to perform complex operations, to provide transformation compensation of original data, or to apply compensated parameters.
• a first aspect of this inability can be associated with the nature of conventional signal processors used with flow cytometry.
• Conventional flow cytometer signal processors often because they are analog, are not capable of dealing with large amounts of signal information, cannot perform operations on low quality signal information, cannot practically accomplish complex transformation operations (such as those which use algebraic expressions or structure), or they perform only reflexive feed back operations rather than serial or multi- variant analysis followed by subsequent parameter compensation.
• a second aspect of this inability can be associated with the infrastructure of conventional data handling.
• conventional infrastructure may not deal with how the streams of information are allocated, aligned, and coordinated.
• Conventional processing of flow cytometer information from occurrences incident to the fluid stream are traditionally handled as isolated feedback loops. As such, it can become increasingly difficult to synchronize various aspects of flow cytometer operation as the number of feed back loops increases.
• these feed back loops may be completely uncoupled.
• stream parameters such as droplet break off location, may be completely uncoupled from the differential analysis of and separation of particles within the fluid stream being compensated.
• a third aspect of this inability may be lack of symmetry reduction in the application of transformed data. Again, analog analysis can prevent or minimize symmetry reduction in the complex analysis of serial occurrences or parallel multivariant analysis. The lack of symmetry reduction or the inability to apply symmetry reduction to analysis terms may increase execution time.
• the present invention uses digital signal processing (DSP) technology to structure information from occurrences incident to flow cytometer operation, and to perform complex transformation, compensation, or analysis operations to achieve this long sought goal.
• DSP digital signal processing
• the present invention discloses a flow cytometer having DSP technology to solve problems associated with high speed serial occurrences, or multiple parameter analysis of occurrences, or both individually or in combination. While specific examples are provided in the context of flow cytometry applications to illustrate the invention, this is not meant to limit the scope of the invention to that field or to applications within flow cytometry. As such, the invention may also have numerous applications in various fields, for example, detection of defects in products as disclosed by United States Patent Nos. 4,074,809 and 4,501,366; field flow fractionation, liquid chromatography, or electrophoresis as disclosed by United States Patent No. 5,503,994; computer tomography, gamma cameras, or time of flight instruments as disclosed by United States Patent No.
• One broad object of an embodiment of the invention can be to convert original signals incident to the environment, the instrument, or a fluid stream, including but not limited to analog signals, to digital signals.
• One aspect of this object of the invention can be to harmonize a plurality of different types of signals into a fresh digitized data stream for processing.
• Another aspect of this object of the invention be to convert otherwise low quality or unusable signal data into usable quality signal data.
• the original signal could be associated with a characteristic or multiple characteristics of single particle, such as a cell, within a fluid stream.
• the original signal could be associated with a characteristic or multiple characteristics of a series of particles within a fluid stream.
• numerous signals may be generated from the sensing of simultaneous occurrences (parallel occurrences) or the sensing of discrete occurrences over time (serial occurrences) that represent one, two, or any number of additional parameters.
• the rate of occurrences sensed may vary between about 10,000 occurrences per second to about 800,000 occurrences per second or more.
• the occurrences may be, as examples, the change in fluid dynamics at the jet or nozzle, the variation of in performance of the equipment itself (such as the change in the baseline electronic signal from a photomultiplier tube), or the variation in performance of equipment due to the change in external conditions such as temperature or pressure.
• the occurrence even when occurring at a high rate, or occurring for a limited duration, or occurring in a sub-optimal manner may be sensed, converted to an original signal, and digitized.
• Another broad object of an embodiment of the invention can be to perform compensation transformation on the original signal to provide compensated parameters.
• One aspect of this object can be to apply compensation transformation to processed data from a first signal incident to a first occurrence and to then apply compensation transformation to processed data from at least one additional signal incident to one or more occurrences to compensate a parameter(s) shared by the first occurrence and by at least one additional occurrence.
• a second aspect of this object can be compensation of parameter(s) that share characteristic(s) so that "cross talk" can be eliminated or minimized. Elimination or minimization of crosstalk provides an increased ability to differentiate a first occurrence from a second or more occurrence(s). Differentiated occurrences may then be assigned to a class, separated, and collected.
• Another object of an embodiment of the invention is to provide hardware or software infrastructure to allocate, align, or coordinate data generated from the above-mentioned original signals.
• One aspect of this object can be to provide multiple signal processors that can operate in parallel to increase the capacity to process signal data.
• the instant invention can utilize at least two but could utilize many parallel signal processors.
• the parallel signal processors could be stand aside hardware, or hardware that can coupled together via ether-net or Internet connections.
• a second aspect of this object of the invention can be to allocate different functions to the various parallel signal processors so as to optimize processing speed.
• a third aspect of this object of the invention can be to use linear assemblers and register usage to enhance parallel operation of and to coordinate the specialized functions performed by at least two signal processors.
• a fourth aspect of this object can be to provide software which optimizes the use of parallel processing of digital code.
• a fifth aspect of this object of the invention can be to apply symmetry reduction to serial transformation operations to reduce processing execution time.
• Another object of an embodiment of the invention can be to perform complex operations on the above-mentioned original signals.
• Complex operations can be operations that were not possible or were not practical prior to the invention due to the speed at which the operations have to be performed in serial or in parallel, the number of parameters involved, the utilization of algebraic expressions or structure, the use of complex numbers to define variables, or the like.
• Each of these aspects can be complex individually or complex in combination.
• Another object of an embodiment of the invention can be to save the original signal in a memory element or memory storage element.
• One aspect of this object can be to save the original signal without altering the original quality or quantity of the original signal. This may be necessary or desirable for quality control concerns or to meet regulatory or statutory requirements.
• Another aspect of this object can be to duplicate the original signal for analysis during flow cytometer operation or to duplicate the signal for future re-analysis.
• Another object of an embodiment of the invention can be to provide software to implement the various applications on DSP technology.
• a first aspect of this object can be to provide exemplary compensation transformation operations. This may include compensation transformation for two way compensation, three way compensation, and so on for higher order compensation sets.
• a second aspect of this object can be to provide exemplary compensation matrices and their various properties.
• a third aspect of this object can be to provide exemplary symmetry reduction in various aspects of the software notation.
• a fourth aspect can be to provide an exemplary program for the subtraction of pairs or groups of fluorescent signals in order to orthogonalize the color sensitivity of each signal.
• Yet another object of an embodiment of the invention can be to provide analog to digital converter compensation of amplified photomultiplier tube (PMT) outputs.
• PMT photomultiplier tube
• emission spectra of fluorescent antibody labels is broadband, they can overlap the passbands of up to eight photomultiplier filters. Therefore, a digitized PMT output from even one antibody label can contain the effects of as many as eight antibody labels. See Shapiro, "Practical Flow Cytometry", pp. 17-19, 163-166 (19 ), hereby inco ⁇ orated by reference herein. This feature allows color sensitivity to be orthogonalized for each signal, and specifically allows for the application in the context of the MoFlo ® flow cytometer.
• Yet another object of an embodiment of the invention can be to provide the ability to latch numerous parameters either simultaneously or interchangeably, and to specifically latch any of the maximum of sixty- four MoFlo ® flow cytometers parameters as inputs.
• Another object of an embodiment of the invention can be to provide cross beam time alignment in order to perform enhanced compensation between a pair of parameters.
• One aspect of this object can be to reduce the apparent inter-beam transition time to not more than
• Another object of an embodiment of the invention can be to provide digital error compensation.
• Digital substraction is attractive because it avoids the problems of signal alignment, however, major digitalization errors can occur. For example, when bright signals are compensated over a large dynamic range digitized errors, which can be visually discemable as a picket-fence coarseness of the compensated population, can occur. Digital error compensation can minimize these errors and hence improve the quality of the digital information.
• Another object of an embodiment of the invention can be to provide log amplifier idealization.
• log amplifiers vary from ideal logarithmic behavior throughout their entire range. For example, some log amplifiers have a 0.4 db variance. That is, for any given input, the ratio of the output signal from a practical log amplifier over the value expected of a perfect logarithmic function is expressed in db as:
• Log amplifier idealization can provide values which more closely approximate the ideal amplifier.
• Another object of an embodiment of the invention can be to provide off-loaded binning.
• the characteristics of , for example, populations of particles can be stored in the memory of a an additional signal processor using binning transformations.
• the statistical characterization of these populations, such as mean, standard deviation, skewness and separation can be sent to a separate processor, thus off-loading this task and hence increasing the performance of the first processor and the separate processor.
• Figure 1 shows a schematic cross sectional view of a flow cytometer embodiment of the invention showing the various features combined.
• FIG. 2 shows a hardware schematic of an embodiment of the invention.
• an enhanced flow cytometer utilizing DSP technology and methods to process raw or original signal information incident to various parameters during operation, including, but not limited to, environmental parameters, instrument parameters, or parameters incident to the particles or cells entrained in a sheath fluid stream allowing for complex assessment, differentiation, assignment, and separation of such particles or cells, even when the flow cytometer is operated at high speed.
• a data acquisition, data transformation, parameter compensation, and compensated parameter utilization system for the differentiation, assignment, and separation of multiple parallel or serial events that can be useful in numerous fields and applications.
• a flow cytometer (1) having a fluid stream source (2) can establish a fluid stream into which particles (3) can be suspended.
• the source of particles (4) can insert the particles from time to time such that at least one particle becomes suspended in and is hydrodynamically focused in the stream.
• An oscillator (5) responsive to the fluid stream perturbs the fluid stream.
• a jet or fluid stream (6) comprised of the fluid stream (2) and the particles (3) can then be established below the tip of the nozzle (7) of the flow cytometer.
• the stream can be established in a steady state condition such that droplets (8) that encapsulate a single particle form and break away from the contiguous part of the stream.
• a stable droplet break-off point can be established below the droplet break-off point (9) .
• a free fall zone (10) can exist below the droplet break-off point (9) .
• This free fall zone embodies the area where the droplets move once they break away from the contiguous part of the stream.
• a sensor (12) such as a laser and receiver in combination (or separately), can be used to monitor the stream for a particle. The sensor can sense an occurrence and generates a signal (15). For example, a coherent beam of light aimed at the fluid stream by the sensor (12) intercepts a particle (3) in the stream (6) and fluorescence or scattered light rays can then be emitted.
• the emitted fluorescence can be captured by the receiver, such as a photomultiplier tube, to generate the signal (15).
• the particle(s) may be differentiated, and assigned to a class.
• a droplet charging location (11) can exist at a point along the free fall zone. Based upon the assignment of the particle, the droplet can be charged positively, negatively, or left uncharged.
• the charged droplets fall in the free fall zone, they can pass through an electrostatic field (12). If the droplets have been charged with a positive or negative charge, an electric field established between these electrostatic plates can deflect the charged droplets such that the trajectory of the deflected droplets (13) and the trajectory of the neutral droplets serves to separate one type of particle class from another. These separated particles can then be collected into a container(s) (14). Furthermore, alternative techniques such as utilizing different quantities of charge can be used to accomplish the assignment and separation of numerous classes of particles. The rate of separating the classes of particles or the sort rate can be at least 1000 per second.
• the sensor (12) can be used to monitor or sense, and then assist in or generate a signal
• the raw or original signal(s) could be associated with a characteristic or multiple characteristics of a single particle (3), which could be a cell, entrained in the fluid stream (2).
• the original signal could be associated with a characteristic or multiple characteristics of a series of particles (3) or cells within the fluid stream (2).
• numerous signals may be generated from the sensing of simultaneous occurrences (parallel occurrences) or the sensing of discrete occurrences over time (serial occurrences) that represent one, two, or any number of additional parameters (at least 64 parameters in the MoFlo ® flow cytometer).
• the rate of occurrences sensed may vary between few per second or could be between about 10,000 occurrences per second to about 800,000 occurrences per second, or even higher.
• the original signal may also represent, as examples, the change in fluid dynamics at the jet or nozzle, the variation in performance of the equipment itself (such as the change in the baseline electronic signal from a photomultiplier tube), or the variation in performance of equipment due to the change in external conditions such as temperature or pressure.
• the parameters could be a variety of aspects incident to the fluorescent emission of fluorenylisothiocyanate (FITC) upon excitation and include pulse width, forward scatter, side scatter, raw FITC information, raw PE, raw PE- CYS, and so forth.
• FITC fluorenylisothiocyanate
• the MoFlo ® system monitors some conventional twelve bit parameters containing pulse width, analog to digital converter (ADC) channel outputs, timer outputs, Look Up Table (LUT) outputs, and the Classifier output.
• ADC analog to digital converter
• LUT Look Up Table
• MoFlo ® users can have need or desire to compute additional parameters which include compensating ADC outputs for the unwanted side effects of broadband fluorescence, computing ratios of ADC channel, and calculating whether ADC parameters fall inside, or outside 3D or higher dimensional regions, and the like.
• the invention employs at least one additional signal processor (17) to apply compensation operations to the processed data from a first signal and to the processed data from a second or more signals.
• the compensated output from the additional signal processor for at least one parameter shared by the signals (or the occurrences which generated the signals) allows enhanced differentiation between the first signal and the second signal based for the compensated parameter(s).
• the compensated data can then be combined into the data handling functions of the first signal processor, for example, and applied to classify and separate the occurrences. Pass Through, Transformation and Return.
• the data emerging from the flow cytometer may exploit at least one additional signal processor, that can for example, be a parallel digital signal processor (17) which may be used simultaneously with a first signal processor.
• the original raw data or a portion of the original raw data from each signal generated by the flow cytometer can be assembled as a table of 32 or more 16 bit data words.
• the first 16 data words could be the raw data outputs from an occurrence, such as fluorescent emissions from excited fluorochromes used as surface or internal markers.
• the first 16 data words may be passed through the additional signal processor and the transformed output may be then presented on the second (or more) 16 data words.
• the final compensated parameters are returned to the first signal processor, combined with the output of the first signal processor, and then presented or displayed. This is often referred to as a pass-through and return digital signal path.
• the numeric data formats for a particular application may have to be matched.
• the raw 12 bit MoFlo ® data can be thought of as a unsigned fixed point integers, in the format 12.0, that is 12 integer bits to the left of the fixed point, and 0 fractional bits to the right of the fixed point. This yields a range of 0x000 (0) to OxFFF (4095).
• the compensated parameter output from the second signal processor (17) may need to be in the same format.
• the internal data manipulations can be changed as required, to perform the required algorithms. Possible internal data formats that could be used are 2's complement, signed integer, signed or unsigned fractional fixed point numbers, or floating point decimal, as examples.
• CPLD/FPGA or digital signal processing Von Neuman or Harvard program, data, and I/O architectures may be used as required to perform algorithms.
• the algorithm and parameter coefficients for the compensated parameters may be changed during instrument operation. If desired, for example in the MoFlo ® system, it should be able to be downloaded at operation time from the system's first computer, for example, through the
• the additional signal processor(s) used in parallel can provide compensated parameters sufficiently fast that the data from numerous signals, channels, or parameters can have compensation transformation performed simultaneously.
• the speed of operation on the first group of 16 data words can occur before the second group of 16 data words becomes available.
• Each data word can pass through the additional signal processor at a rate of at least every 150 nanoseconds. Consequently, the additional signal processor can perform all operations to which it has been assigned for 16 data words within a maximum period of about 2410 nanoseconds.
• compensation transformation operations on the data from a signal(s) can provide compensated parameters to differentiate occurrences during flow cytometer operation.
• the additional signal processors can perform compensation transformation operations for selected parameters even when the occurrences which are being differentiated have a rate of at least 10,000 per second or up to 800,000 occurrences per second.
• the additional signal processor(s) could apply compensation transformation operations to occurrences having lower rates as well.
• the compensated parameters generated by the additional signal processor(s) are then returned to the first signal processor.
• the first signal processor can handle data for different tasks than the additional signal processors.
• the first signal processor can perform the task of data management and display while the additional signal processors are performing, among other others, compensation transformation functions on the original signals.
• the separation of the tasks of data management and display and parameter compensation transformation may be an essential requirement to achieve accurate and reliable function.
• compensation transformation including complex operations, can be performed on the emission spectra of flourescent antibody labels which overlaps the passbands of eight PMT filters.
• the compensation transformation operations can take the following form, and while this may be a preferred arrangement, a great variety of alternative embodiments are possible.
• Two way compensation Two linear signals from 0 to 1000 mv converted to a log signal in such a fashion that the log and linear voltages are related:
• V , log A.log(V , Ill /V th ) (1)
• V 2 , og A.log(V 2 l ⁇ n /V th ) (2)
• V/ th is normally 1 millivolt. This formula ensures that an input from 1 millivolt to 10000 millivolts will produce a log signal from 0 to 10000 millivolts with 2.5 volts per decade.
• a compensated parameter is a parameter with cross-talk subtracted out between two parameters. This is given by:
• V , lm V th exp (V 1 , og /A) (5)
• V 2 l ⁇ n V th exp (V 2 log /A) (6)
• linear values may be then applied to (3) and (4) above and converted to log by reapplication of (1) and (2). In practice, this calculation will be performed on digital values whose linear range is 0 to 4095 (post digitization) and where the threshold value is 4095. IX 0000.0.
• v lc n v m (x - c rv ⁇ n rv m (x - c rv ⁇ m rv m (7)
• V 3c l ⁇ n V 3 l ⁇ n (l - C 3i rV 1 l ⁇ n /V 3 l ⁇ n (l - C 32 rV 2 l ⁇ n /V 3 lake n (9)
• N-color compensation can be deconstructed to N-1 2D lookups.
• the 3 -color compensated output when followed through from anti-log and back to log may look like this:
• V lc log LUT(V'log, V 2 , og ) - V 3 Iog )
• the compensation matrix may be as follows:
• Poc Po" c o ⁇ • e(P ⁇ -po)- C o2-e(p 2 -Po)-Co3-e(p 3 -Po)-Co4-e(p4-Po)- C o6-e(p6-Po)-Co7- e (P7-Po) (10)
• P 2c -c 2 o-e(Po-P 2 )-C 2 . -e(PrP 2 )+p 2 c 23 e(p 3 -p 2 )-C 24 .e( ⁇ 4 -p 2 -c 2S e(p 5 -p 2 )-c 26 .e(p 6 -p 2 )-c 27 .e(p 7 -p 2 ) (12)
• P3c - C 30- e (P ⁇ -P3)- C 31-e(P 1 -P3)-C 3 2-e(p2-p3) + P3- C 4-e(P 4 -P3)-C35e(P5-P3)- C 36- e (P6-p3)- C 37- e (p7-P3) (13)
• P 4C -c 4 o-e(Po-P 4 )-c 4 ⁇ -e(p 1 -p 4 )-C 42 .e(p 2 -p 4 )-c 43 .e(p 3 -p 4 )+p 4 -c 45 e(p 5 -p 4 )-c 46 .e(p 6 -p 4 )-c 47 .e(p 7 -p 4 ) (14)
• P6c - c 6o- o-P6)-C6 1 -e(PrP6)-C62-e(P2-P6)-C63-e(P 3 -p6)-c 6 4 e (p4-P6)- c 65-e(P5-p6) + P6- c 67-e(P7-P6)
• P7c - C 70-e(Po-P7)- C 7, - l-P7)- C 72- 2-p7)- C 73-e(P3-P7)- C 74e(P4-P7)- C 75- e (P 5 -p7)- C 76- e (P5-P7) + P7 (17)
• the functions e(P j -p k ) may range from exp(-4095/A) to exp(4095/A) since p n may be always positive and in the range 0 to 4095. This is a range from 1/10000 to 10000 which is an eight decade range.
• the calculation of e(p j -p k ) should be done with a 16 bit map to preserve memory space, but the values in the lower ranges less than 1.0 are badly represented. This means that calculation accuracy cannot be maintained across all mapped values of e(p k -p k ).
• the MoFlo ® parameter bus runs at 150 ns per frame word, thus the number of MoFlo ® data words is:
• the last compensation parameter is in slot 10.
• the output needs to be ready at data word 16.
• the calculation matrix cannot be done as each MoFlo ® parameter comes across because the off-diagonal elements et ⁇ j -p,,) may be mixtures of all parameters.
• the pipelining and parallel architecture of the DSP can allow substantial reduction of this calculation time.
• Symmetry reductions can be made on this set in order to reduce execution time.
• the equations above can be multiplied by e(p n ) and the diagonal terms moved to the left side
• Occurrences separated in time can be, in the flow cytometer context, for example, different original or raw signals generated for the same particle as it moves through the various flow cytometer processes which as above-described involve entrainment into a fluid stream, excitation of bound fluorochrome, assignment to a class, and separation of particles to the assigned classes. Occurrences separated in time can also involve a particle labeled with several different fluorochromes with each type of fluorochrome excited at different points in time.
• occurrences separated in time could be a series of discrete occurrences each monitored for the same parameter, such as a fluorescent emission from a series of labeled cells, or it could be a single occurrence monitored at discrete periods in time, such as the characteristics of a flourescent emission as it decays.
• occurrences separated in time could be a series of discrete occurrences each monitored for the same parameter, such as a fluorescent emission from a series of labeled cells, or it could be a single occurrence monitored at discrete periods in time, such as the characteristics of a flourescent emission as it decays.
• numerous other examples could be provided which have occurrences separated in time.
• the spatial separation of these occurrences leads to original signal output which is separated in time.
• the use of additional signal processor(s) using pass through, compensation transformation, and return can remove this temporal separation.
• Compensation transformation on the original or raw signals can remove "cross-talk" between the same or different parameters which are incident to the same or different occurrences, different occurrences incident to the same parameters, or "cross talk” incident to. As described above, the "cross talk" between different types of fluorochrome emission was compensated. Compensation transformation may allow the raw original fluorescent signals, or numerous other types of signals, to be compensated so that the resulting compensated parameter has the cross-talk accurately removed and blank reference signals correctly positioned. This may be particularly relevant to other types of applications such as the detection of defects in products as disclosed by United States Patent No. 4, 074, 809 and 4,501,366; field flow fractionation, liquid chromatography, or electrophoresis as disclosed by United States Patent No.
• Compensation transformation for multiple color compensation can take the format presented above and allow for at least 8 color compensation embodied by a 64 element matrix of operations.
• the transformation can operate on linear or logarithmic format data. Naturally, as explained higher order set can be used providing for N-color compensation.
• Ratios between two signals over time can be an important measurement in the study of cell kinetics.
• the original signals can be compensated such that the ratio can be used to provide a measure of absolute differences between the signals.
• calcium release can be an important measurement for the study of cell kinetics.
• a ratio of two fluorescent emission signals can be required to provide a measure of calcium release.
• These fluorescent emission signals can have compensation transformation applied to provide compensated fluorescent emission signals for comparison in the appropriate time frame required to maintain accuracy. Multiple ratios can also be performed.
• Time can also be a parameter essential for kinetic measurements and can be supplied by the on-board clock.
• the on-board clock can have a time range from microseconds to years allowing full flexibility in time-stamping data streams.
• Flow cytometers depend on the stability of various parameters, including, but not limited to, environmental parameters, instrument parameters, occurrence parameters to analyze and define the mean and width of particle populations. Unfortunately, these parameters can be in continuous dynamic instability. Stability can be controlled by compensation transformation of the original signals from these various parameters. Alternately, compensation transformation can track the drift in these parameters.
• Compensation transformation of original signal information can allow for the selection of parameters to resolve or differentiate sub-population, to select the level of resolution to be maintained between individuals of sub- populations, to select the thresholds for assignment and separation of individuals from sub- populations, to allow for continuous differentiation and assignment of individuals from sub- populations to various classes, to track sub-populations as parameters drift, to assess the purity of pools of separated individuals without re- analysis, among other applications.
• two dimensional, three dimensional, or higher dimensional populations of particles can be differentiated and assigned to various sub-populations and multi-dimension regions can be used to separate the sub-populations when using the invention.
• This provides a powerful and direct method of multi-dimensional sub-population separation that has been previously unavailable on flow cytometers, and on other types of instrument, and in other fields of application .
• sub-population identification involves closely overlapping sub- populations can be enumerated by dynamically characterizing the overlap using compensation transformations that may be designed to detect the proportion of overlaps.
• the exact proportions, mean, width and separation of multi-featured sub-populations can also be characterized with the invention.
• Extensive populations of particles with small sub- populations of interest can be focused upon and held in dynamic amplification or focus through transformation compensation of amplification parameters such that the sub- populations of interest can be defined, located, analyzed, and separated. Without transformation compensation, such accurate delineation may not be possible.
• particles with various population(s)/sub- populations of interest can be screened and regions of interest can be created which delineate these populations.
• These regions can be automatically assigned to the sorting electronics of a flow cytometer so that real-time physical separation of the particles of interest can be sorted.
• This automation process can be important when flow cytometry is used to separate high volumes of certain types of cells for culturing, transfecting, insemination, biochemical recombination, protein expression, or the like.
• Populations of particles can be stored in the memory of the addition signal processor(s) using binning transformations.
• the statistical characterization of these populations, such as mean, standard deviation, skewness and separation can be returned to the first signal processor, that can be a workstation for display, storage, or retrieval of data.
• the first signal processor that can be a workstation for display, storage, or retrieval of data.
• Attachment A can preserve the raw signal data in a memory storage element. Cost considerations often exclude this feature on an analog systems. Saving raw or original signal data also conforms to Good Manufacturing Practice in that the original signal data can be retrieved if the transformed data has been incorrectly manipulated. By saving the original signal data and duplicating original signal data for further processing, elements of the original raw signal data that may be lost by digital 'roofing' or 'flooring' can be maintained. This can allow original signal retrieval and data backtracking for FDA requirements and for signal re-analysis.
• the additional signal processor (17) can be located internal to or external to the core of the instrument.
• a minimum data memory size of 56 kilowords of 12 bits or wider may be required for each compensation transformation operation (based on the example above).
• a minimum I/O memory space of TBD kilowords may also be required.
• Various CPLD/FPGA or digital signal processing Von Neuman and Harvard program, data, and I/O architectures, or the like, may be used to perform compensation transformation algorithms, such as those specified above.
• Additional processors (17) serve to increase the parallelism of the operations, thus allowing transformations at hitherto unachievable speeds. This increased power allows operations that are algebraic as well as approximately transcendental. Transcendental operations can be considered those requiring an infinite number of steps. However extremely high processing rates can provide approximations to the infinite that are practicable and indistinguishable from an exact computation.
• the basic concepts of the present invention may be embodied in a variety of ways. It involves both signal processing techniques as well as devices to accomplish the appropriate signal processing.
• the processing techniques are disclosed as part of the results shown to be achieved by the various devices described and as steps which are inherent to utilization. They are simply the natural result of utilizing the devices as intended and described.
• devices are disclosed, it should be understood that these not only accomplish certain methods but also can be varied in a number of ways. Importantly, as to all of the foregoing, all of these facets should be understood to be encompassed by this disclosure.
• each of the various elements of the invention and claims may also be achieved in a variety of manners.
• This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these.
• the words for each element may be expressed by equivalent apparatus terms or method terms ⁇ even if only the function or result is the same.
• Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled.
• each of the processing devices or subroutines as herein disclosed and described ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods, iv) those alternative designs which accomplish each of the functions shown as are disclosed and described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the elements disclosed, xi) processes performed with the aid of or on a computer as described throughout the above discussion, xii) a programmable apparatus as described throughout the above discussion, xiii) a digitally

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