AU781985B2 - Transiently dynamic flow cytometer analysis system - Google Patents

Description

WO 01/28700 PCT/US00/41372 TRANSIENTLY DYNAMIC FLOW CYTOMETER ANALYSIS SYSTEM I. TECHNICAL FIELD Specifically, 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.

II. BACKGROUND 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.

This type of analysis requires uniform conditions within the jet, very precise timing, and consistent comparative parameters incident to the entrained substances to separate such substances accurately. In addition, there is a coincident commercial and public sector demand for higher speed flow cytometry, the need to differentiate substances based on more complex and multiple parameter analysis, and for higher purity separation(s). Unfortunately, variation in equipment operation, sheath fluid stream dynamics, or observed particle characteristics still exists and are exacerbated by increasing the speed at which entrained substances are carried in the jet. As such, there is a need to compensate for such variations to provide for accurate analysis and separation of the substances entrained in the sheath fluid stream.

An overview of some attempts tn iinderstnd and react to fluid stream and droplet dynamics can be seen in United States Patents Nos. 4317520, 4318480, 4318481, 4318482, 4318483, and 4325483, each hereby incorporated by reference herein. As these explain, traditionally the approach has been to assess the signals and act directly upon such information. Some of the practical problems which have also been recognized is the fact that only a limited amount of space and time exists within which to conduct sensing and analysis.

004637076 As Japanese Patent 2024535 also recognizes with respect to the sensing system alone, it may be desirable to have an optical system which is as small as possible.

As can be understood, a substantial problem can be that the data generated from an occurrence must be sensed and reacted upon in an extremely short period of time. Given the speed of microprocessors and the like, this might, at first glance, appear to be readily achievable.

The challenge for this unique flow cytometry situation is that original or raw signal data can be sub-optimal and even unusable. As such, if it is to be used, it must be further processed in order to accomplish further analysis or decision making. This processing can be complex and can require more processing speed and power than is available not just with typical commercial systems, but even with today's highest-speed computer systems. Further, as the desire for higher processing frequencies is pursued, problems can be compounded. An example of the extremes to which speed has been taken is shown in United States Patent No. 4361400, hereby incorporated by reference herein, where droplet formation frequencies in the range of 300 to 800 kilohertz had been achieved. Most practical droplet flow cytometers operate in the range of 10 to 50 kHz.

Although speed of analysis problems have been known for years, prior to the present invention it has apparently been an accepted attitude that digital analysis in the flow cytometry context could not be achieved. This invention proves this expectation to be untrue. As a result of the present invention, droplet formation speeds in the 50-100-200 or higher kHz ranges are now possible with adequate data compensation and the like.

At any of these speeds, however, there appears to have been an expectation that analog analysis was the only practical way to achieve analysis of and to compensate for fluid dynamics, particle characteristics, equipment variance, and the like. To some degree, these expectations have been so prevalent that quality control, good manufacturing practices, regulatory approval, and other concerns have been set aside, diminished, or even compromised. The previously existing technology governing the practices of those in this field.

9 :Another significant problem associated with conventional analysis and compensation of variables in flow cytometry can be the preservation of original signal data from an WO 01/28700 PCT/US00/41372 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 reanalysis 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, 0o to provide transformation compensation of original data, or to apply compensated parameters.

Conventional analysis can be limited by the amount of information that can be processed and returned in between serial events which can occur at a rates of at least 10,000 per second.

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 multivariant analysis followed by subsequent parameter compensation.

A second aspect of this inability can be associated with the infrastructure of conventional data handling. In part, 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 crn become increasingly dificult to synchronize various aspects of flow cytometer operation as the number of feed back loops increases. Moreover, these feed back loops may be completely uncoupled. For example, 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.

004637076 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.

As mentioned above, there has been a long felt but unsatisfied need for apparatus and methods which permit complex signal transformation, and use of compensated parameters resulting from complex signal transformation, real time analysis using compensated parameters, or storage of original signal data generated incident to the fluid stream, instrument variance, or environmental variance. The present invention addresses each of the above-mentioned problems with a practical solution. To some extent, it is apparent that solutions have not been achieved because those skilled in the art seem to have taken a direction which was away from the technical direction pursued in the present invention. This may have been the result of the fact that those skilled in the art did not truly appreciate the nature of the problem or it may have been the result of the fact that those skilled in the art were misled by some of the presumptions and assumptions with respect to the type of systems which could be considered. The present invention uses digital signal processing (DSP) technology to structure information from occurrences incident to flow cytometer operation, and to perform complex transformation, 1o: 20 compensation, or analysis operations to achieve this long sought goal.

III. DISCLOSURE OF THE INVENTION One aspect of the present invention provides a flow cytometer comprising a fluid stream, a sensor responsive to an occurrence incident to the environment, the flow cytometer or the fluid stream, at least one signal generator coupled to the sensor, a first signal processor to perform :25 operations on signal data from the signal generator, a second signal processor to perform operations on the signal data, compensated parameter output from the second signal processor returned to the first signal processor, and a particle differentiation element responsive to the compensated parameter output.

Another aspect of the present invention provides a method of flow cytometry, comprising the steps of establishing a fluid stream, perturbing the fluid stream, sensing an occurrence incident to the fluid stream, generating a signal from the occurrence, processing the signal using a first signal processor, processing the signal using at least one additional signal processor, 4 004637076 combining output from the first signal processor and the at least one additional signal processor and applying the combined output to classify the occurrence.

In one form of the invention, the signal generator provides a first data signal and at least one additional data signal, wherein the signal processor is responsive to the first data signal and to the at least one additional data signal such that a transformation operation is applied to the first data signal and, further, the particle differentiation element is responsive to signal comparisons produced by a signal comparison element.

In another form of the invention, the signal generator provides signal data incident to a first occurrence and a second occurrence, wherein the compensated parameter is shared by the first and second occurrences, and wherein a first transformation operation is applied to at least a portion of the signal data relating to the first occurrence and a second transformation operation is applied to at least a portion of the data relating to the second occurrence.

In at least one embodiment, 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 o• S 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. 5,880,457, each of the above-mentioned patents are hereby incorporated by reference herein. It should be understood that the basic concepts of the invention may be applied not only to the area of flow cytometry but may apply to 25 each of the above mentioned fields, or to other fields where the detection and analysis of small S differences in parameters, such as photo-generated signal between serial occurrences having high incident light flux, or serial occurrences generating data concerning multiple parameters, or occurrences that generate a high number of signals in a short period of time, may be necessary or desired. Moreover, it should be understood that the invention can be divided into a number of embodiments which may be combined in various permutations and combinations.

Naturally, as a result of these several different and potentially independent aspects of the invention, the objects of the invention are quite varied.

004637076 In at least one embodiment, the invention enables the conversion of 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 embodiment(s) can be to harmonize a plurality of different types of signals into a fresh digitized data stream for processing. Another aspect of this embodiment(s) can be to convert otherwise low quality or unusable signal data into usable quality signal data. In this regard, the original si..al could be associated wit; a characteristic or multiple characteristics of single particle, such as a cell, within a fluid stream. Alternately, the original signal could be associated with a characteristic or multiple characteristics of a series of particles within a fluid stream. As such, 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 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. As to each, 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.

:20 In at least one embodiment, the invention enables the performance of compensation transformation on the original signal to provide compensated parameters. One aspect of this embodiment(s) 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 nroces..d dta from at least one additional signal incident to one or more occurrences to compensate a "-25 parameter(s) shared by the first occurrence and by at least one additional occurrence. A second ee.: aspect of this embodiment(s) can be compensation of parameter(s) that share characteristic(s) so .o that "cross talk" can be eliminated or minimized. Elimination or minimization of cross talk 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.

In at least one embodiment, the invention provides hardware or software infrastructure to allocate, align, or coordinate data generated from the above-mentioned original signals. One aspect of this embodiment(s) can be to provide multiple signal processors that can operate in 6 004637076 parallel to increase the capacity to process signal data. Embodiments of the 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 be coupled together via ether-net or Internet connections. A second aspect of this embodiment(s) can be to allocate different functions to the various parallel signal processors so as to optimize processing speed. A third aspect of this embodiment(s) can be to use linear assemblers and register usage to ehnce,, parallel operation of and to coordinate the specialized functions performed by at least two signal processors. A fourth aspect of this embodiment(s) can be to provide software which optimizes the use of parallel processing of digital code. A fifth aspect of this embodiment(s) of the invention can be to apply symmetry reduction to serial transformation operations to reduce processing execution time.

In at least one embodiment, the invention enables the performance of 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.

06 In at least one embodiment, the invention enables the saving of the original signal in a memory element or memory storage element. One aspect of this embodiment(s) can be to save 20 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 embodiment(s) can be to duplicate the original signal for analysis during flow cytometer operation or to duplicate the signal for future re-analysis.

0% o o 0In at least one embodiment, the invention provides software to implement the various 25 applications on DSP technology. A first aspect of this embodiment(s) 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 embodiment can be to provide exemplary compensation matrices and their various properties. A third aspect of this embodiment can be to provide exemplary symmetry reduction in various aspects of the software notation. A fourth aspect of this embodiment 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.

7 004637076 In at least one embodiment, the invention provides analog to digital converter compensation of amplified photomultiplier tube (PMT) outputs. Since emission spectra of fluorescent antibody labels are 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, 163-165 (1995), hereby incorporated by reference herein. This feature aillows coor sensitivity to be orthogonalized for each signal, and specifically allows for the application in the context of a flow cytometer such as those sold under the trademark MOFLO®.

In at least one embodiment, the invention provides 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.

In at least one embodiment, the invention provides cross beam time alignment in order to perform enhanced compensation between a pair of parameters. One aspect of this embodiment(s) can be to reduce the apparent inter-beam transition time to not more than 1 part in 3000 or to a compensated beam to beam "time jitter" of not more than one nanosecond, which appears to be beyond the practical capability of analog circuit design.

In at least one embodiment, the invention provides digital error compensation. Digital subtraction 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 20 dynamic range digitized errors, which can be visually discerable as a picket-fence coarseness of the compensated population, can occur. Digital error compensation can minimize these errors 0 and hence improve the quality of the digital information.

In at least one embodiment, invention provides log amplifier idealization. Typically log amplifiers vary from ideal logarithmic behavior throughout their entire range. For example, some :25 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: Error 0.4 db 201ogl0 (Vout/Videal) 004637076 Log amplifier idealization can provide values which more closely approximate the ideal amplifier.

In at least one embodiment, the invention provides off-loaded binning. The characteristics of, for example, populations of particles can be stored in the memory of an additional signal processor using binning transformations. The statistical characterization of these populations, such as mean, standard deviation, skewness and separation can e ent to a separate processor, thus off-loading this task and hence increasing the performance of the first processor and the separate processor.

IV. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic cross sectional view of a flow cytometer embodiment of the invention showing the various features combined.

Figure 2 shows a hardware schematic of an embodiment of the invention.

V. MODE FOR CARRYING OUT THE INVENTION Specifically, an enhanced flow cytometer utilizing DSP technology and methods to :15 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. Generally, a data acquisition, data transformation, *20 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.

In discussing these aspects of the invention some references may be made to MOFLO® (a trademark of Cytomation, Inc.) flow cytometer systems and SUMMIT® (also a trademark of Cytomation, Inc.) capabilities for such systems. Each of these systems represent state-of-the-art flow cytometry capabilities which are not only the fastest practical flow cytometer systems, but they also are well known to those of ordinary skill in the art.

004637076 Referring now to Figure 1, a preferred embodiment of the invention can be seen in detail.

A flow cytometer having a fluid stream source can establish a fluid stream into which particles can be suspended. The source of particles 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 responsive to the fluid stream perturbs the fluid stream. A jet or fluid stream comprised of the fluid stroam and the particles can then be established below the tip of the nozzle of the flow cytometer. The stream can be established in a steady state condition such that droplets that encapsulate a single particle form and break away from the contiguous part of the stream. When the stream is established in this steady state fashion, a stable droplet break-off point can be established. Below the droplet break-off point a free fall zone can exist. This free fall zone embodies the area where the droplets move once they break away from the contiguous part of the stream. A sensor 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 For example, a coherent beam of light aimed at the fluid stream by the sensor (12) intercepts a particle in the stream 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 Based upon analysis of the signal generated by the sensor from the fluorescent occurrence, the particle(s) may be differentiated, and assigned to a class. A droplet charging location (11) can exist at a point along 20 the free fall zone. Based upon the assignment of the particle, the droplet can be charged positively, negatively, or left uncharged.

As the charged droplets fall in the free fall zone, they can pass through an electrostatic field If the dronlets have heen chnrpeA with a nnsitive nr npontivp charoP an pip-trr fipid established between these electrostatic plates can deflect the charged droplets such that the 25 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) 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 incident to a variety of parameters (16) related to the operation of a flow cytometer or numerous other instruments (used individually or in combination). As described above, the raw 004637076 or original signal(s) could be associated with a characteristic or multiple characteristics of a single particle which could be a cell, entrained in the fluid stream Alternately, the original signal could be associated with a characteristic or multiple characteristics of a series of particles or cells within the fluid stream As further mentioned above, numerous signals may be generated from the sensing of simultaneous occurrences (parallel occurrences) or the sensing of discrete occurrences over time (serial occurrences) that represen 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. Specifically, as shown in Figure 1 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 phycoerythnin, raw PE (raw phycoerythrin), and so forth. Naturally, numerous other parameters could be also be monitored and these specific examples are meant to be used as a guide rather than an inclusive list.

The MYFLO® flow cytometer system, for example, 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. MOFLO® flow cytometer ystm ulsPrs can haveP need or desire to compute addtijal pairtcrs i il 2. 5 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. To expand the capability of instruments such as the MOFLO® system, other types of flow cytometer systems, or other types of instruments, 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.

This may occur in parallel or simultaneous with the data processing of a first signal processor 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 004637076 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. Again referring to Figure 1, and as mentioned above, 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 passthrough and return digital signal path.

Naturally, the numeric data formats for a particular application may have to be matched.

For example the raw 12 bit MOFLO® flow cytometer data can be thought of as a unsigned fixed *o 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 to OxFFF (4095).

S The compensated parameter output from the second signal processor (17) may need to be in the 20 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 oO integer, signed or unsigned fractional fixed point numbers, or floating point decimal, as examples. Various CPLD/FPGA (Complex Programmable Logic Devices/Field Programmable 4 Gate Arrays) 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 1: coefficients for the compensated parameters may be changed during instrument operation. If ~desired, for example in the MOFLO® flow cytometer system, it should be able to be downloaded at operation time from the system's first computer, for example, through the MFIO RevB Control Word Bus, using the same programming convention.

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 12 004637076 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 every150 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.

As such, compensation transformation operations on the data from a siral(s) can provide compensated parameters to differentiate occurrences during flow cytometer operation. For such real time operation of a flow cytometer or other instrument, the additional signal processor(s) 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. Naturally, 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. As such, the first signal processor can handle data for different tasks than the additional signal processors. In one embodiment of the invention, 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. Thus, 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.

S. S :20 As but one example of using the invention, with or without additional signal processor(s), compensation transformation, including complex operations, can be performed on the emission spectra of fluorescent 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.

T @o :25 Two-way compensation 0* Two linear signals from 0 to I 000mV converted to a log signal in such a fashion that the log and linear voltages are related: A.log(V',,/Vj A.log(V',,/V) where A 10000/log(10000N/V 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: 0 6 6600 9 S@ 0O

S

6@

OS@@

0 S S

S

0r 60 6 0

SOS.

S

S

0e5* VCia V'ii (1 Cl 2

)^V

2 1 ifVll, V2,= (1 C 2 ,/V2 1 In order to recover the V 1 and V2,. from the log values, the inverse functions of(1) and (2) may be evaluated: 15 V, exp V,,i V, exp (V, 2

/A)

These linear values may be then applied to and above and converted to log by reapplication of(l) and 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 Jl0000.0.

Three-way compensation Mathematically this is the same process except that the formulae for the compensation set is: Vi VI 1

C

12 IV 2 1 //V'l(1 =2 i V 1 l- C 2 l)AVi.N/ 2 jjj1 C 23

)AV

3 1

./V

2 ti. v3% -3i( C3/tV3/1I -C 2

)AV

2 1 u/V 3 1 in...

and so on for higher order compensation sets.

The lookup tables could be used for N-color compensation in the following way. Following this note on the transformation it is clear that N-color compensation can be deconstructed to N-i 2D lookups. For example, the 3-color compensated output when followed through from anti-log and back to log may look like this: VIlg= VI log- exp(V, 0

V',

0 ,)-log(l c, 2 exp(V 3

V'

1 Iog0(1 _C 13 0~in e -it- I%;t-,Itgc alld th-e 'Las L Lerin of this expression it is equivalent to: 15 Vic log LUT(V'log, V'10,) LUT(V',.g, Applyina the Transformatin The following notation convention is used to describe and eight-by-eight compensation matrix: p,,c where n =0 to 7 are the compensated outputs WO 01/28700 PCTUSOO/41372 p. are the input log signals where n 0 to 7 are the compensation coefficients -A*log(l -Cj where Cjkare the fractional compensation values ranging from 999 to .999 e(pn -pm) exp((p. p~j/A) A 4095.0/log (10000) 444.6 The compensation matrix may be as follows: pO-C 0 1 -p 0 )-c 02 .eP 2 -p)-C 3 e-p)-co.e 4

-P

0 )-co.e(p 6 -p 0

)-C

0 7 e(p 7

-P

0 -c 10 .eQ, 0 -p 1 )+p 1

-C

12 .e(p 2 -Pl)-c 3 .eP 3

-P

1 )-c 14 .e(p 4 -Pl)-Cl 5 e(p 5 -p 1

)-C

16 .e(p 6

-P

1 7 .e(p 7

-P

1 (11) -c- 20 .e(p 0 -p 2 )-c 21 .e(p 1 -p 2

)+P

2 -C2e(p 3 -p 2 )-c 2 4 e4P 4 -p 2 25 c(p 5

-P

2

)-C

26 e(p 6 -p 2 )-c 2 7.e(p 7 -p 2 (12) -c 30 .e(p 0 -p 3 )-c 31 .e(p 1 -p 3

)-C

3 2 .e(P 2 -p 3 )+p 3

-C

34 .e(P 4

-P

3

)-C

35 e(P 5 -p 3

)-C

36 .e(p 6 -p3)-c 37 -e(p 7 -p 3 (13)

-C

4 0 .e(P 0 -p 4 )-c 41 .e(p 1 -p 4 )-c 42 .e(p 2 -p4j-C 43 .e(~p 3 -P)+p 4

-C

45 e(p 5 -p4j-C 46 e(p 6 -p 4 )-c 47 .e(P 7 -p 4 (14) 'i-u ri )2'r I-52-W2 rS/ -53 '3P5I4 s) 4-5k's) -56 -kY6PSJ "57-XP7 P6c -C60.c(p 0

-P

6

)-C

61 .e(p,-P 6

)-C

62 .e(p 2 -p 6

)-C

63 .e(P 3 -p 6 ,)-c6e(p 4 -p 6

)-C

65 e(p-P 6

)+P

6 -c 67 e(, 7 -p 6 (16) WO 01/28700 PCT/USOO/41372 P7c -70.e(po-p 7 )-c 7 1 c 2e(2-P7)-c 7 3 .e(p 3 -p 7 )-c 7 4 e(p 4

-P

7 )-c 75 .e(ps-P 7 )-c 6 .e(p 5

-P

7 )+p 7 (17) Note that the c, are positive or negative and the parameters from which the others are subtracted are along the diagonal of the matrix.

P tpertic There is symmetry around the diagonal in that the e(pj-p) terms one side of the diagonal are the inverse of those on the other. However this is not a useful symmetry since division is a time consuming operation on an integer arithmetic DSP device.

The functions e(p,-pk) may range from exp(-4095/A) to exp(4095/A) since p, 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. In order to do fast integer arithmetic, preferably the calculation of e(p-p) should be done with a 16 bit map to preserve memory space, but the values in the lower ranges less than are badly represented. This means that calculation accuracy cannot be maintained across all mapped values of e(p,-Pk).

It may be necessary to have two maps, one for positive and the other for negative arguments of eO in order to maintain accuracy.

Given these constraints, we can calculate the number of operations which may be needed to resolve this matrix.

Operations Speed (clocks) Clocks Pointer Loads 4 4 16 (2 maps, p. pointer, c, pointer Sum Initialization 8 1 8 Loads ofp. 8 4 32 Subtracted Pairs 28 1 28 Mappings 56 4 224 Loads of c 1 56 4 224 Multiplies 56 2 112 Subtractions 56 1 56 Stores 8 1 8 Total 280 708 The 6201 DSP runs at a clock cycle of 5 ns. Thus, this calculation for non-optimized execution is 5*708 3540 ns. The MOFLO® flow cytometer system parameter bus runs at 150 ns per frame word, thus the number of MOFLO® flow cytometer system datawords is: 3540/150 23.6 The last compensation parameter is slot 10. The output needs to be ready at data word 16. The calcuation matrix cannot be done as each MOFLO® flow cytometer system parameter comes across because the off-diagonal elements e(pj-pk) 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(pj and the diagonal terms moved to the left side p0,-p).e(pj) =0 -c 0 1 .e(p 1 .e(p 2 )-ca .ep 3 )-cO.Ie(p 4 )Cse(p)-c 6 e(P)-C 7 S2(pj.-pj.ee= -pj) 0--eO _(P)CI-(17 (p0 1 0 e 0 +0 -c 3 .e(p )-c5.e(p 4 )-ce(ce(pCe p) (p 2 -c3 0 .e(p 0 1 e(p 1 +0 C4cC. -c3 e(pj-c,,.eP (p-p 4 )-e(P 4 -c 40 .e(p)-c 41 .e(p)-c 4 2 .e(p)-C 4 3 +0 -C 45 .e(p 5 )-c4.6e(p 6 )-c 47 .e(p 7 (ps.-p 5 ).e(p 5 =-c 50 .e(p 0 )-c 5 e(p 1 )-c 52 .e(P 2 )-c 53 .e(P 3 )-c51.e(p 4 +0 -C 56 .e(P6j-C 57 .e(p 7 (p,-pj.e(pj 62-e Pp)-3e(c3-c64e 465e PS) -c 7 e(P 7

(P

7 -c 0 .e(p)-c 7 7 e(p 3 )-c 4 .c(pj-c.e(p)-c 6 0 Pointer loads Sum initialization Loads of pn Mappings Loads of cj Multiplies Subtractions Post NORM Post SHL Pointer loads Post loads Post remap Post multiples Post SHIEFT ADD Post SHR Post adds 20 Stores Operation 4 8 8 8 32 64 64 8 8 2 8 8 8 8 8 8 8 Speed (clocks) 2 1 4 4 4 2 1 1 1 2 4 4 2 1 1 1 Clocks 8 8 32 32 128 128 64 8 8 4 32 32 16 8 8 8 8 Total 532 The execution time for this matrix is 2660 ns which is MOFLO® flow cytometer system frame words 2660/150 17.7 MOFLO® flow cytometer system data words.

Using the linear assembler and optimization of register usage to enhanced parallel operation can yield the parallel code set out in Attachment A, hereby incorporated by reference herein. This program, and the above-described example is not meant to limit the invention to specific hardware, software, algorithms, applications, or arrangements, but is provided as a guide in making and using the invention which may take the form of various embodiments. Particular embodiments of the invention, in the flow cytomerry context or otherwise, can be as follows.

In certain applications, occurrences can be separated in time. 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. Again 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 fluorescent emission as it decays. Of course, 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. In some cases this will enable certain application which were heretofore not possible, such as the use of multiple separate lasers to excite multiple fluorochromes over 20 time, in other cases it will allow the original signals to have compensation transformation S* applied and more accurate differentiations made between occurrences even during operation of the instrument. Operations such as this which are have a_ In ,lerancc fr i JiiLr often cannot be performed using an analog arrangement because of the difficulty of removing the temporal separation with analog circuitry.

25 In certain applications "cross talk" between the same or different parameters can occur.

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.

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 004637076 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. 5,503,994; computer tomoraphy. gamma cameras, or time of flight instruments as disclosed by United States Patent No. 5,880,457; or flow cytometry as disclosed by United States Patent No.

5,135,759 with respect to bright fluorescent values, each hereby incorporated by reference herein. This type of compensation transformation can be performed on numerous channels simultaneously, at least 8 channels in the above-described example, and provides orthogonalized data which can be returned to the first signal processor.

Certain applications require multiple color compensation. 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.

Certain applications require analysis of parameter kinetics or ratios. Rations 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 20 differences between the signals. For example, 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 onboard clock. The on-board clock can have a time range from microseconds to years allowing full flexibility in time-stamping data streams.

*o Certain applications require differentiation of and tracking of sub-populations. 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 Scontinuous dynamic instai 'tability canh- bc controlled b 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 subpopulations, to select the thresholds for assignment and separation of individuals from subpopulations, to allow for continuous differentiation and assignment of individuals from subpopulations 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.

In this regard, 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 instruments, and in other fields of application.

20 Another aspect, of sub-population identification involves closely overlapping sub- -populations can be enumerated by dynamically charactenzi2 Ih era AMYUsing cumpensation transformnations 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 subpopulations of interest can be focused upon and held in dynamic amplification or focus through transformation compensation of amplification parameters such that the subpopulations of interest can be defined, located, analyzed, and separated. Without transformation compensation, such accurate delineation may not be possible.

In applications using flow cytomerry, particles with various population(s)/subpopulations of interest can be screened and regions of interest can be created which delieate 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.

automation process can be important when flow Wy-- tr is se tosprt I volumes of certain types of cell 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. Thus off-loading this task to the additional signal processor can increase the performance of the workstation.

The meth od described above and detailed in Attachment A can preserve the raw signal is data in a memory storage element. Cost considerations often exclude this feature on an analog sysems Saingrawor riginal 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 *20 'flooring' can be maintained. This can allow original signal retrieval and data backtracking .for FDA requirements and for signal re-analysis.

Now referring to Figure 2, a preferred embodiment of the hardware with respect to an application of the invention with the MOFLO®0 flow cytometer is shown. As can be understood, 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 1/0 memory space of TBD kilowords may also be required. Various CPLD/FPGA or digital signal processing Von Neuman and Harvard program, data, and 1/0 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.

As can be easily understood from the foregoing, the basic concepts of the, "resent invention may be embodied in a variety of ways. It involves both signal processing uiques as well as devices to accomplish the appropriate signal processing. In this application, 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. In addition, while some 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.

The discussion included in this application is intended to serve as a basic *.:description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternntives nre im cT. 15t wl a ~y not fuily explain the generic nature of the invention and may not explicitly show how each feature or :::element can actually be representative of a broader fuinction or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in functionally-oriented terminology, each aspect of the finction is :25 accomplished by a device, subroutine, or program. Apparatus claims may not only be included for the devices described, but also method or process claims may be included to Saddress the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of the claims which now be included.

Further, 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. Particularly, it s should be understood that as the disclosure relates to elements of the invention, 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. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action.

Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, as but one examnple, the disclosure of a "processor" should be understood to encompass disclosure of the act of "processing" whether explicitly discussed or not and, conversely, were there only disclosure of the act of "processing", such a disclosure should be understood to encompass disclosure of a "processor" and even a means for "processing". Such changes and alternative terms are to be understood to be explicitly included in the description.

.Additionally, the various combinations and permutations of all elements or :20 applications can be created and presented. All can be done to optimize the design or performance in a specific application.

Any acts of law, statutes, regulations, or rules mentioned in this application for patent: :or patents, publications, or other references mentioned in this application for patent are hereby incorporated by reference. Specifically, United States Patent Application No. 60/160,719 is 25 hereby incorporated by reference herein including any figures or attachments, and each of references in the following table of references are hereby incorporated by reference.

V WO 01/28700 PCT/USOO/41372 I. U.S. PATENT DOCUMENTS DOCUMENT DATE NAME CLASS SUBCLASS FILING NO. DATE 3299354 12/17/67 Hogg 207 582 07/05/62 3661460 05/09/72 Elking et al. 356 36 08/28/70 3710933 0/16/73 Fulwyler et al 209 3 12/23/71 3761941 09/25/73 Robertson 346 I 10/13/72 3810010 05/07/74 Thoem 324 71 1/27/72 3826364 07/30/74 Bonner et al 209 3 05/22/72 3833796 11/03/74 Fetneretal 235 151.3 10/13/71 3960449 07/01/76 Carleton et al 356 103 06/05/75 3963606 06/15/76 Hogg 209 3 06/03/74 3973196 08/03/76 Hogg 324 71 06/05/75 4014611 03/29/77 Simpson et al 356 72 04/30/75 4070617 01/24/78 Kachel et al 324 71 08/03/76 4162282 07/24/79 Fulwyler et al 264 9 04/22/76 4230558 10/28/80 Fulwyler 209 3.1 10/2/78 4302166 11/24/81 Fulwyleretal 425 6 03/15/79 4317520 03/02/82 Lombardo et al 209 3.1 08/20/79 4318480 03/09/82 Lombardo et al 209 3.1 08/20/79 4318481 03/09/82 Lombardo et al 209 3.1 08/20/79 4318482 03/09/82 Barry et al 209 3.1 08/20/79 4318483 03/09/82 Lombardo et al 209 3.1 08/20/79 4325483 04/20/82 Lombardo et al 209 3.1 08/20/79 4341471 07/27/82 Hogg et al 356 343 01/02/79 4350410 09/21/82 Minott 350 170 10/08/80 4361400 11/30/82 Gray etal 356 23 11/26/80 4395676 07/26/83 Hollinger ct al 324 71.4 11/24/80 4400764 08/23/83 Kenyon 362 263 05/19/81 4487320 12/11/84 Auer 209 3.1 11/03/80 4498766 02/12/85 Unterleitner 356 73 03/25/82 5274 0S7,o toiiingcret ai 209 3.1 12/02/81 4523809 06/18/85 Toboada et al 350 163 08/04/83 4538733 11/03/85 Hoffman 209 3.1 10/14/83 4598408 07/01/86 O'Keefe 372 94 10/22/84 4600302 07/15/86 Sage, Jr. 356 39 03/26/84 4631483 12/23/86 Pronietal 324 71.4 02/01/84 4673288 06/16/87 Thomas ct al 356 72 11/07/84 WO 01/28700 PCT7USOO/41372 4691829 09108/87 Auer 209 3.1 12/06/84 4702598 10/27/87 Bohmer 356 343 02/25/85 4744090 05/10/88 Freiberg 372 94 07/08/85 4758729 07/19/88 Monnin 250 560 08/28/87 4794086 01/27/88 Kaspcr et al 436 36 11/25/85 4818103 04/04/89 'Thomas et a 356 72 01/20/87 4831385 05/16/89 Archcr ctal 346 1.1 10/14/87 4845025 07/04/89 Lary etal 435 2 1 !0/87 4877965 10-31-89 Dandliker et al 250 458.1 07-01-85 4942305 07/17/90 Sommer 250 574 05/12/89 4981580 01/01/91 Auer 209 3.1 05/01/89 4983038 01/08/91 Ohki et al 356 246 04/07/88 5005981 04/09/91 Schulte et al 366 219 09/08/89 5007732 04/16/91 Ohkietal 356 73 04/18/88 5030002 07/09/91 North, Jr. 356 73 08/11/89 5034613 07-23-91 Denket al 250 458.1 11-14-89 5079959 01/14/92 Miyakeetal 73 864.85 09/08/89 5098657 03/24/92 Blackford et al 422 73 08/07/89 5101978 04/07/92 Marcus 209 3.1 11/27/89 5127729 07/07/92 Oetliker et al 356 317 10/15/86 5144224 09/01/92 Larsen 324 71.4 04/01/91 5150313 09/22/92 Van den Engh et al 364 569 04/12/90 5159397 10/27/92 Kosaka et al 356 73 09/05/9 I 5159403 10/27/92 Kosaka 356 243 03/19/91 5167926 12/01/92 Kimura ctal 422 67 09/11/90 5180065 01/19/93 Tougeetal 209 577 10/11/90 5182617 01/26/93 Yoneyama et al 356 440 06/29/90 5199576 04/06/93 Corio ct al 209 564 04/05/91 5215376 06/01/93 Schulte etal 366 348 03/09/92 5247339 09/21/93 Ogino 356 73 09/05/91 5259593 11/09/93 Orme et al 266 78 04/16/92 5260764 11/09/93 355 73 65/29 5298967 03/29/94 Wells 356 336 06/02/92 5359907 11/01/94 Baker et al 73 865.5 11/12/92 5370842 12/06/94 Miyazaki ct al 422 82.06 11/20/92 5412466 05/02/95 Ogino 356 246 05/22/92 5452054 09/19/95 Dewaetal 355 67 11/21/94 5466572 11/14/95 Sasaki, et al 435 2 0425/94 WO 01/28700 PCT/USOO/41372 5466572 11/14/95 Sasaki, et al 435 2 04/25/94 5467189 11/14/95 Kreikebaum et al 356 336 01/12/95 5483469 01/09/96 Van den Engh et al 364 555 08/02/93 5523573 06-04-96 Hanninen et al 250 459.1 12-28-94 5558998 09/24/96 Hammond, et al 435 6 06/05/95 5596401 01/21/97 Kusuzawa 356 23 09/14/94 5601235 02/11/97 Booker et al 239 4 11/15/94 S 02039 02-11-97 Van de Enigh 436 164 iO-14-94 5602349 02-11-97 Van den Engh 73 864.85 10-14-94 5641457 07/24/97 Vardanega, et al 422 82.01 04/25/95 5643796 07/01/97 Van den Engh et al 436 50 10/14/4 5650847 07/22/97 Maltsev et al 356 336 06/14/95 5672880 09-30-97 Kain 250 458.1 03-15-96 5675401 10/07/97 Wangler ct al 355 67 06/15/95 5700692 12/23/97 Sweet 436 50 09/27/94 5707808 01/13/98 Roslanicc et al 435 6 04/15/96 5726364 03-10-98 Van Den Engh 73 864.85 02-10-97 5759767 06-02-98 Lakowicz ct al 435 4 10-11-96 5777732 06-07-98 Hanninen ct al 356 318 04-27-95 5786560 07-28-98 Tatah et al 219 121.77 06-13-97 5796112 08-18-98 Ichie 250 458.1 08-09-96 5815262 09-29-98 Schrof et al 356 318 08-21-96 5835262 11-10-98 [kctaki et al 359 352 12-28-95 5912257 06-15-99 Prasad et al 514 356 09-05-96 II. FOREIGN PATENT DOCUMENTS DOCUMENT DATE COUNTRY CLASS SUBCLASS NO. EP0468100AI 01/29/92 Europe G01N15 14 EP0160201A2 11/06/85 Europe G01N15 14 JP4126064 27/04/92 Japan A23P1 08 JP4126065 04/27/92 Japan A23PI 12 JP4126066 04/27/92 Japan CI2MI 02 JP4126079 04/27/92 Japan C12N9 48 WO 01/28700 WOOI/8700PCT/USOO/4 1372 JP4 126080 04/27192 Japan C12149 JP4126081 04/27/92 Japan CI 2N 15 02 JP61139747 06/27/86 Japan GO IN21 53 JP2024535 01/26/90 Japan (301NO15 14 SU1056008 11/23/83 Soviet Union GOIN021 24 JP61159135 07118/86 Japan GOIN21 17 FR2699678-Al 12123/92 France GOIN21 64 SU!260778-A! 0%310e us GOIN21 64 EP 0781985 A2 07-02-97 Germany (Karis et al.) DE19549015 03-04-97 Germany 21 WO099/44037 02126/99 English GOIN 6 Ill. OTHER DOCUMENTS (including Author, Title, Date, Pertinent Pages, Etc.) An Historical Review of the Development of Flow Cytometers and Sorters, Melamed et al, 1979, pp.

3-9 Axicon; Journal of the Optical Society of America; Vol. 44, Eastman Kodak Company, Hawk-Eyc Works, Rochester, NY, 09/10/53, pp. 592-597 Ceruzzi, "History of Modem Computing", MIT Press, Reference to Non-von Neumann.

D.L. Garner, et al; "Quantification of the X- and Y- Chromosome-Bcaring Spermatozoa of Domestic Animals by Flow Cytometry', Biology of Reproduction 28, pgs. 312-321, (1983) Denk, eta] (1995). Two-photon molecular excitation in laser scanning microscopy. Handbook of Biological Confocal Microscopy. J.B. Pawley, ed., Plenum Press, New York. pp 444-458.

Flow Cytometry: Instrumentation and Data Analysis, Van Dilla et al. "Overview of Flow Cytomerry: Instrumcrntation and Data Analysis" by Martin Van Dilla, 1985, pp. 1 -8 LwecA.ohsn"SxreectobyFlow Cytomet ntuettotndDt nlss a il Septio of. XEs) andoY Chromorsomed barigmplermndin, base onDNA Difne al. 1 eview pepp. F7t7.Dv 99,7,ps.83-0 FlJw Skoe-Haensod Cetl "rHingh Effirc Ey*Oetoo Flow Cytometer,"Th Jouna ofisonr en ycitry Vol. 2519.79p.7478,177,S WO 01/28700 PCT/USOO/41372 Manni, Jeff; (1996). Two-Photon Excitation Expands The Capabilities of Laser-Scanning Microscopy, Biophotonics International, pp 44-52 Piston, et al (1994). Two-photon-excitation fluorescence imaging of three-dimensional calcium ion activity. APPLIED OPTICS 33:662-669 Piston, et al. (1995). Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in-situ cornea with two-photon excitation laser scanning microscopy. J OF MICROSCOPY 178:20-27 Shapiro, H. "Practical Flow Cytometry", Third Edition, John Wiley Sons, Inc., Publication.

Williams, R.M. et al. (1944). Two photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J. 8:804-813.

"An Intrroduction to Flow Cytometry", pp 5-7 and pp 33-42 and page In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. However, as to each of the above, to the extent that such information or statements incorporated by reference might be considered inconsistent with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicant(s).

In addition, unless the context requires otherwise, it should be understood that the term "comprise" or variations such as "comprises" or "comprising", are intended to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Such terms should be interpreted in their most expansive form so as to afford the applicant the broadest coverage legally permissib!e in countries such as Australia and the like.

Thus, the applicant(s) should be understood to have support to claim at least: i) 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 WO 01/28700 PCT/USOO/41372 of these devices and methods, iv) those alternative designs which accomplish each of the functions shown as are disclosed and described, v) those alterative 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 readable memory encoded with data to direct a processor comprising means or elements which function as described throughout the above discussion, xiv) a computer configured as herein disclosed and described, xv) individual or combined subroutines and programs as herein disclosed and described, xvi) the related methods disclosed and described, xvii) similar, equivalent, and even implicit variations of each of these systems and methods, xviii) those alternative designs which accomplish each of the functions shown as are disclosed and described, xix) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, xx) each programmable feature, component, and step shown as separate and independent inventions, and xxi) the various combinations and permutations of each of the above.

Claims (17)

1. A method of flow cytometry, comprising the steps of: a. establishing a fluid stream; b. perturbing said fluid stream; c. sensing an occurrence incident to said fluid stream; d. generating a signal from said occurrence; e. processing said signal using a first signal processor; f. processing said signal using at least one additional signal processor in parallel with said first signal processor; g. utilizing at least a portion of code by said first signal processor and said at least one additional signal processor; h. combining output from said first signal processor and said at least one additional signal processor; and h. applying said combined output to classify said occurrence.

2. A method of low cytumelry as described in claim i, wherein said step ot utilizing at least a portion of code by said first signal processor and said at least one additional processor comprises utilizing at least a portion of digitized code by said first signal processor and said at least one additional signal processor.

3. A method of flow cytometry as described in claim 2, further comprising the steps of: performing compensation transformation on said signal; and generating a compensated signal.

4. A method of flow cytometry as described in claim 3, wherein said step of performing compensation transformation on said signal comprises compensating a single parameter. 004637076 A method of flow cytometry as described in claim 4, wherein said step of compensating a signal parameter comprises compensating an analog signal.

6. A method of flow cytometry as described in claim 3, wherein said step of compensating an analog signal comprises minimizing variations selected from the group consisting of phase, and shape.

7. A method of flow cytometry as described in claim 3, wherein said step of performing compensation transformation on said signal comprises compensating at least two different parameters.

8. A method of flow cytometry as described in claim 7, wherein said step of compensating at least two different parameters comprises minimizing characteristics shared by at least two different parameters.

9. A method of flow cytometry as described in claim 8, wherein said step of minimizing characteristics shared by said at least two different parameters comprises reducing spectrum overlap.

10. A method of flow cytometry as described in claim 3, wherein said step of performing compensation transformation comprises applying algebraic operations on said signal. oooo go. o•*

11. A method of flow cytometry as described in claim 10, further comprising applying a o* compensation matrix.

12. A method of flow cytometry as described in claim 11, further comprising the step of :0 minimizing execution time of performing compensation transformation on said signal by utilizing symmetry reductions in said compensation matrix.

13. A method of flow cytometry as described in claim 3, wherein said step of performing compensation transformation on said signal comprises performing compensation transformation S: on a plurality of signals generated at a rate of 50,000 occurrences per second or greater.

14. A method of flow cytometry as described in claim 1, further comprising the step of binning information in said at least one additional signal processor. 004637076 A method of flow cytometry as described in claim 1, further comprising the step of saving original data from said signal in a memory storage element; and retrieving said original data from said signal saved in said memory storage element substantially without alteration.

16. A method of flow cytometry as described in claim 1, further comprising the steps of: a. duplicating original data from said signal; b. processing said duplicate signal of said original data; and c. analyzing said occurrence using said processed duplicate signal.

17. A flow cytometer as comprising: a. a fluid stream 0 b. a sensor responsive to an occurrence; 0 0 0% 0 0000 0 0 0..0 0*.0 2 0 c. at least one signal generator coupled to said sensor; d. a first signal processor to perform operations on signal data; e. a second signal processor to perform operations on said signal data; f. compensated parameter output from said second signal processor returned to said first signal processor; and g. a differentiation element responsive to said compensated parameter output.

18. A flow cytometer as described in 17 further comprising a. a particle assignment element; b. a particle separator; and c. at least one container in which separated particles are collected. 004637076%

19. A method of flow cytometry including the steps substantially as herein described. A flow cytometer substantially as hereinbefore described with reference to the accompanying drawings. Dated 27 April 2005 Freehills Patent Trade Mark Attorneys Patent Trade Mark Attorneys for the Applicant: DakoCytomation Colorado, Inc.