HK1148587B - Methods for controlling one or more parameters of a flow cytometer type measurement system - Google Patents

Description

Method for controlling one or more parameters of a flow cytometer type measurement system

The patent application of the invention is a divisional application of the invention patent application with the international application number of PCT/US2004/026225, the international application date of 2004, 08/13, and the application number of 200480023013.5, entitled "method for controlling one or more parameters of a flow cytometer type measurement system", which enters the national phase of China.

Technical Field

The present invention relates generally to methods for controlling one or more parameters of a flow cytometer type measurement system. Certain embodiments relate to methods that include changing one or more parameters of a flow cytometer type measurement system in real time based on parameter monitoring.

Background

The following description and examples are not admitted to be prior art by virtue of their inclusion in this section.

Generally, flow cytometers provide a measurement of the fluorescence intensity of a laser-excited polystyrene bead or cell as it passes linearly through a flow cell. However, flow cytometry may also be used to provide measurements of one or more characteristics of other particles. Some systems are configured for measuring the level of light scattered by the particles at 90 ° or 180 ° to the excitation source, two or more fluorescence measurements being used to determine the classification, i.e. the "identity" of the particle, further fluorescence measurements, known as "indicators" (reporters), are often used to quantify the chemical reactions of interest. Each fluorescence measurement is made at a different wavelength.

As the measurement capabilities of flow cytometer type measurement instruments have improved, the field of application in which flow cytometers can provide useful measurements has increased dramatically. For example, flow cytometers have become increasingly helpful in providing data for applications such as biological assays (e.g., displacement or competition assays, non-competition assays, enzyme assays), nucleic acid analysis, and combinatorial chemistry. In particular, flow cytometers are becoming increasingly popular due to the speed at which assays are performed, particularly in comparison to other assay methods, such as conventional enzyme-linked immunosorbent assay "ELISA" formats.

In a typical environment, to ensure accurate and reliable assay results, calibration of a flow cytometer is performed as one or more initial steps in preparing the instrument for proper use and measurement. Furthermore, there is no guarantee as to the source of variation between samples unless the fluorescence channels of each flow cytometer are calibrated to perform the same reading. If a complete and robust calibration method is not used, it is likely that the same instrument will give different readings for the same sample on different days. Similarly, if any two instruments are not guaranteed to give the same results even if they are correctly set up, although flow cytometry may be a better measure of identifying and distinguishing between cells in a sample, its use as a medical instrument may be less useful.

Thus, many different methods for calibrating flow cytometers have been developed. At first, effective studies have been made to develop a method of reducing the involvement degree of the operator at the time of calibration, thereby improving the accuracy of calibration. This result largely automates many steps in the calibration of flow cytometers. Furthermore, effective research has been conducted to otherwise improve the accuracy of calibration. For example, this effort has led to advances in calibration, such as using calibration standards with uniform and constant characteristics. In particular, since the properties of biological samples may change over time, the biological calibration standards used in flow cytometry are essentially replaced by integrated calibration standards (e.g., polymeric microspheres or particles) that have more stable properties. Furthermore, typically, the calibration microspheres have properties (e.g., dimensions, volume, surface properties, particle size properties, refractive index, fluorescence, etc.) that are substantially similar (i.e., as close as possible) to those of the test microspheres. It is believed that such calibration microspheres improve the accuracy of the flow cytometer by calibrating values as close as possible to those expected in the test.

Efforts to improve the calibration of flow cytometers have also led to an increase in the number of parameters of the flow cytometers that are relevant for calibration. For example, the laser excitation, detectors, and electrons of a flow cytometer measurement system may change over time, thereby affecting the final measurement. Therefore, these parameters of the flow cytometer, and in some cases other parameters, are often associated with the calibration method.

Other more difficult to control parameters also affect flow cytometry measurements. Sample speed is one such parameter. An example of a method for measuring the velocity of a sample is described in U.S. patent No. 6,532,061 to Ortyn et al, which is incorporated by reference as if fully set forth herein. In this method, the object is entrained in a liquid flow flowing through a sensing or measuring space. In each of these embodiments, a grating having a substantially uniform pitch is used to modulate light received from a moving object. The modulated light is converted to an electrical signal, which is digitized and then subjected to a Fast Fourier Transform (FFT) to determine the velocity of the object. However, the method and system of Ortyn et al for measuring sample velocity has some drawbacks. For example, these methods require a rather complex grating and software. Furthermore, gratings can be quite expensive due to the precision required of the grating and the complexity of its manufacture. Also, optical distortions of the detected light, such as due to moving objects, may result in less accurate sample velocity measurements.

However, the largest error in flow cytometry measurements is typically caused by temperature variations. Furthermore, it has been found that the existing available calibration methods do not adequately account for the effect of temperature changes on measurements made by flow cytometers. For example, the method and system described by Ortyn et al does not take into account temperature variations, despite its attempt to calibrate some parameters, and how these variations will affect the measurements of the flow cytometer. Thus, although there are many different possible calibration methods, further improvements can be made to these methods by more accurately taking into account temperature variations in different flow cytometer measurements or in individual flow cytometer measurements.

It would therefore be beneficial to develop methods for controlling at least the major error factors of flow cytometer measurement systems that can be integrated to produce a real-time calibration scheme.

Disclosure of Invention

As detailed previously, the most significant error factor in most flow cytometers in general is temperature variation. Since temperature can be a measurable quantity and the physical mechanism behind its effect is known, it is possible to reduce or even completely eliminate the most critical one of these error sources.

Several causes of measurement errors and real-time correction techniques for these causes of measurement errors have been identified. Furthermore, a method has been created for real-time fine tuning using calibration microspheres that can be identified as having a diameter slightly different from the analyte that may be contained in the microsphere sample mixture. Additional features of the fine tuning process include real-time identification of system health status, correction of non-linearities in one or more channels, and/or significant extension of the useful indicator dynamic range of the flow cytometer measurement system. The described embodiments can be used to compensate for system variations that are primarily caused by temperature, thereby extending the calibration range of operation.

Furthermore, it is noted that several different embodiments of methods for controlling one or more parameters of a flow cytometer measurement system are described herein. It is to be understood that each method can be used and performed separately. In addition, two or more methods may be used in combination, depending on, for example, the variability of various components of the measurement system and/or the desired accuracy of the measurement system.

One embodiment of the invention is directed to a method of controlling one or more parameters of a flow cytometer type measurement system. The method includes monitoring one or more parameters of a flow cytometer type measurement system as the measurement system performs measurements of sample microspheres. The method also includes changing the one or more parameters in real-time based on the monitoring.

In one embodiment, monitoring one or more parameters may include monitoring one or more parameters using measurements of calibration microspheres. The calibration microspheres have a diameter that is different (e.g., slightly smaller) than the diameter of the sample microspheres. In certain embodiments, the one or more parameters may include an output signal generated by a detector of the measurement system. The output signal is responsive to light scattered by the sample microspheres.

In another embodiment, monitoring one or more parameters may include monitoring one or more parameters using measurements of calibration microspheres. The calibration microspheres have a different (e.g., slightly smaller) diameter than the sample microspheres, and at least some of the calibration microspheres have different spectral addresses (spectral addresses). In one such embodiment, the one or more parameters include a dynamic range of the measurement system. In another embodiment, changing the parameters may include extending a dynamic range of one or more channels of the measurement system. In yet another embodiment, the one or more parameters may include a measure of system health. The measure of system health may include the health of the classification channel, the health of the indicator channel, or a combination thereof. In certain embodiments, the one or more parameters may include linearity in the measurement of the sample microspheres. In such an embodiment, the measurements may include measurements of the classification channel, measurements of the indicator channel, or a combination thereof. In another such embodiment, the changing of the parameter includes approximately correcting for any non-linearity in the measurement.

In certain embodiments, the parameter may comprise a parameter of an avalanche photodiode of the measurement system. In one such embodiment, the method may also include using empirically obtained data to determine a correction factor to be used in changing the parameter. In another embodiment, the parameter may comprise a parameter of a photomultiplier tube of the measurement system.

In yet another embodiment, the parameter may comprise a velocity of the sample microspheres. In one such embodiment, monitoring the parameter may include monitoring a temperature of a liquid in which the sample microspheres are disposed and determining a velocity of the sample microspheres based on the temperature. In certain embodiments, the method may further comprise calibrating the one or more parameters prior to measuring the sample microspheres. Each of the embodiments of the foregoing method may include any other step described herein.

Another embodiment relates to a different method of controlling one or more parameters of a flow cytometer type measurement system. The method includes monitoring a temperature proximate to a flow cytometer type measurement system. The method also includes using the empirical data to change a bias voltage of an avalanche photodiode of the measurement system in response to temperature to substantially correct for variations in gain of the avalanche photodiode due to temperature.

In one embodiment, the method further comprises generating the empirical data by applying a substantially constant light level to the avalanche photodiode at one or more temperatures and recording the current output of the avalanche photodiode for a plurality of bias voltages at the one or more temperatures. In another embodiment, the change to the parameter is made before the measurement system makes the sample measurement. In one such embodiment, the bias voltage may remain substantially constant during the sample measurement. In a different embodiment, the monitoring and changing of the parameters is performed in real time.

In some embodiments, the method may further comprise varying the bias voltage of the avalanche photodiode while the calibration microspheres that emit light of known intensity are measured by the measurement system until a predetermined signal level is obtained from the avalanche photodiode. In one such embodiment, the method may also include determining a respective relative current of the avalanche photodiode at a reverse bias voltage, a bias voltage at a predetermined signal level, and a temperature. Embodiments of the method may also include determining the bias voltage using the corresponding relative current, temperature, reverse bias voltage, and empirical data. Each embodiment of the foregoing method may include any other step described herein.

Another embodiment relates to yet another method of controlling one or more parameters of a flow cytometer type measurement system. The method includes monitoring a temperature proximate to a flow cytometer type measurement system. The method also includes varying an output signal of a photomultiplier tube of the measurement system in response to temperature using a characteristic curve of the photomultiplier tube to substantially correct for gain variations in the output signal of the photomultiplier tube. The gain of the photomultiplier tube varies approximately linearly in response to temperature. In certain embodiments, the photomultiplier tube is part of an indicator channel of a measurement system. In another embodiment, the characteristic curve of the photomultiplier tube varies with the detection wavelength and the cathode structure of the photomultiplier tube. Each embodiment of the method described herein before may include any other step described herein.

Another embodiment is directed to another embodiment of a method of controlling one or more parameters of a flow cytometer type measurement system. The method includes setting a photomultiplier tube voltage of the measurement system to a first value and a second value. The method also includes measuring an output current of the photomultiplier tube at a first value and a second value. In addition, the method includes determining a calibration voltage of the photomultiplier tube based on a logarithm of the first and second values relative to a logarithm of the output current at the first and second values. The method also includes applying the calibration voltage to the photomultiplier tube. The method also includes testing the photomultiplier tube to determine whether one or more parameters of the photomultiplier tube are within a predetermined tolerance. Each embodiment of the foregoing method may include any other step described herein.

Yet another embodiment relates to another method of controlling one or more parameters of a flow cytometer type measurement system. The method includes determining a calibration voltage of a probe of the measurement system using successive approximation. The method also includes applying the calibration voltage to the detector. In one embodiment, the detector may comprise an avalanche photodiode. In a different embodiment, the detector may comprise a photomultiplier tube.

In one embodiment, the method includes comparing the calibration voltage to a breakdown voltage of the detector and repeating the determining of the calibration voltage if the calibration voltage exceeds the breakdown voltage. A different embodiment of the method includes collecting and processing detector samples to determine detector signal levels. In one such embodiment, the method may include comparing the detector signal level to a calibration target signal level, reducing the bias voltage of the detector if the detector signal level is above the calibration target signal level, and repeating the determination of the calibration voltage. In another such embodiment, the method may include comparing the detector signal level to a calibration target signal level and, if the detector signal level is not within a predetermined range of the calibration target signal level, repeating the determination of the calibration voltage until all desired detector voltage levels have been tried. Each embodiment of the foregoing method may include any other step described herein.

Yet another embodiment relates to a different method of controlling one or more parameters of a flow cytometer type measurement system. The method includes monitoring a temperature of a liquid that will flow through a flow cytometer type measurement system. Sample microspheres are placed in the liquid. The method also includes determining a velocity of sample microspheres in the measurement system based on the viscosity of the liquid at the temperature.

In one embodiment, the method further comprises determining a length of time that one of the sample microspheres will be present in a detection window of the measurement system based on the velocity. In certain embodiments, the method may include determining a length of time for one of the sample microspheres to move from one detection window to another detection window of the measurement system based on the velocity. In another embodiment, the method may include determining when one of the sample microspheres is present in a detection window of the measurement system based on the velocity. In yet another embodiment, the method includes controlling a sampling interval of one or more detection windows of the measurement system to compensate for the velocity.

In yet another embodiment, monitoring the parameter and determining the velocity are performed prior to the measurement system taking a measurement of the sample microspheres. In certain embodiments, the method may include determining one or more characteristics of an output signal of the measurement system as a function of the velocity. In one such embodiment, the method includes using the correction factor to correct for errors in the output signal due to velocity. The correction factor is determined using empirical measurements. In another embodiment, the measurement system is configured to maintain the pressure of the liquid substantially constant during the measurement of the sample microspheres.

In one embodiment, determining the velocity includes determining the velocity from a table, a Poiseuille equation, or from a predetermined value of the velocity corresponding to the temperature. In some embodiments of the parts of speech, the method may further comprise controlling the pressure of the liquid during measurement of the sample microspheres according to the velocity. Each embodiment of the foregoing method may include any other step described herein.

A different embodiment relates to another method of controlling one or more parameters of a flow cytometer type measurement system. The method includes measuring a time for a microsphere to move from a first detection window of a flow cytometer type measurement system to a second detection window of the measurement system. The method further includes varying the applied pressure of the measurement system such that the time is substantially constant. In one embodiment, the time is an average time. The microspheres may be sample microspheres or calibration microspheres. Measuring the time may include measuring light scattered by the microspheres in the first and second detection windows. In another embodiment, measuring the time may include measuring light scattered by the microspheres at the first and second detection windows with a single detector. Light scattered by the microspheres in the first and second detection windows may be directed by a beam splitter to said one detector. The method may be operated in real time or non-real time. Each embodiment of the foregoing method may include any other step described herein.

Yet another embodiment relates to a different method of controlling one or more parameters of a flow cytometer type measurement system. The method includes measuring an average time for a microsphere to move from a first detection window of a flow cytometer type measurement system to a second detection window of the measurement system. The microspheres may include sample microspheres, calibration microspheres, or both. The method also includes comparing the average time to a reference time taken for the reference microsphere to move from the first detection window to the second detection window. Further, the method includes changing the applied pressure of the measurement system if the difference between the average time and the reference time is greater than a predetermined value.

In one embodiment, varying the applied pressure comprises: the applied pressure is increased if the average time is greater than the reference time. Alternatively, the applied pressure is decreased if the average time is less than the reference time. In some embodiments, the predetermined value is selected to compensate for known time varying mechanisms of the measurement system. The method may be operated in real time or non-real time. Each embodiment of the foregoing method may include any other step described herein.

Drawings

Other objects and advantages of the invention will become apparent upon reading the following detailed description and upon reference to the drawings in which:

FIG. 1 is a schematic diagram illustrating a measurement system that may be used to perform the methods described herein.

Fig. 2 is a graph illustrating a plurality of bias curves showing the response of an APD with a reverse bias voltage (V60) of 130 volts as a function of temperature.

Fig. 3 is a graph depicting the response of various PMTs as a function of temperature.

Fig. 4 is a graph illustrating the logarithm of PMT gain as a function of the logarithm of PMT bias voltage.

FIG. 5 is a flow chart describing one embodiment of a method for controlling one or more parameters of a flow cytometer type measurement system.

FIG. 6 is a schematic diagram illustrating a cross-sectional view of one embodiment of a portion of a measurement system that may be used to perform at least one of the methods described herein.

FIG. 7 depicts a pulse burst (i.e., scattered light measured at different times) that may be measured in one embodiment of the methods described herein.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

Detailed Description

Several different embodiments of methods for controlling one or more parameters of a flow cytometer type measurement system will be described herein. As mentioned above, each method can be used and carried out separately. Further, two or more methods may be used or performed in combination, depending on, for example, the variability of various components in the measurement system and/or the desired accuracy of the measurement system.

Although the embodiments described herein refer to microspheres or polystyrene beads, it should be understood that the measurement systems and methods may also be used with microparticles, gold nanoparticles, beads, microbeads (microbeads), latex particles, latex beads, fluorescent particles, colored beads, and cells (cells). The microspheres can serve as carriers for molecular reactions. Examples of suitable microspheres, beads, and particles are described in U.S. Pat. No. 5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al, U.S. Pat. No. 6,057,107 to Fulton, U.S. Pat. No. 6,268,222 to Chandler et al, U.S. Pat. No. 6,449,562 to Chandler et al, U.S. Pat. No. 6,514,295 to Chandler et al, U.S. Pat. No. 6,524,793 to Chandler et al, and U.S. Pat. No. 6,528,165 to Chandler et al, all of which are incorporated herein by reference in their entirety. The measurement systems and methods described herein may be used with any of the microspheres, beads, and particles described in these patents. In addition, microspheres for flow cytometry are available from manufacturers such as Luminax Corp, Otton, Tex. The terms "bead" and "microsphere" are used interchangeably herein.

FIG. 1 illustrates one example of a measurement system that may be used to implement the methods described herein. In particular, one or more parameters of the measurement system depicted in FIG. 1 may be determined, monitored, varied, and/or controlled according to the methods described herein. It should be noted that the figures described herein are not drawn to scale. In particular, the dimensions of some of the elements in the figures may be exaggerated to emphasize characteristics of the elements. For clarity of illustration, certain elements of the measurement system are not included in the figures.

In fig. 1, the measurement system is shown along a plane through a cross-section of the cuvette 12 through which the microspheres flow. In one example, the cuvette may be a standard quartz cuvette, such as that used in a standard flow cytometer. However, any other suitable type of viewing or transport chamber may be used to transport the sample to be analyzed. The measurement system includes a light source 14. Light source 14 may include any suitable light source known in the art, such as a laser. The light source may be configured to emit light having one or more wavelengths, such as blue or green light. Light source 14 may be configured to illuminate the microspheres as they flow through the cuvette. The illumination may cause the microspheres to fluoresce with one or more wavelengths or wavelength bands. In certain embodiments, the system may include one or more lenses (not shown) configured to focus light from the light source onto the microspheres or onto the flow path. The system may also comprise more than one light source. In one embodiment, the light sources may be configured to illuminate the microspheres with light having different wavelengths or wavelength bands (e.g., blue light and green light). In certain embodiments, the light sources may be configured to illuminate the microspheres in different directions.

Light scattered forward from the microspheres may be directed to a detection system 16 by a folding mirror 18 or another suitable light guide assembly. Alternatively, detection system 16 may be placed directly in the path of the forward scattered light. As such, a folding mirror or other light guide component may not be included in the system. In one embodiment, the forward scattered light may be light scattered by the microspheres at an angle of about 180 ° to the direction of illumination of the light source 14, as shown in FIG. 1. The forward scattered light may not be at exactly 180 ° to the direction of illumination so that incident light from the light source does not hit the photosensitive surface of the detection system. For example, the forward scattered light may be light scattered by the microspheres at an angle less than or greater than 180 ° from the direction of illumination (e.g., light scattered at about 170 °, 175 °, 185 °, or 190 °).

Light scattered by the microspheres at an angle of about 90 ° to the direction of illumination may also be collected (collected). In one embodiment, the scattered light may be split into more than one beam by one or more beam splitters or dichroic mirrors. For example, light scattered at an angle of about 90 ° to the direction of illumination may be split into two different beams of light by the beam splitter 20. The two different beams of light may be further split into four different beams of light by splitters 22 and 24. Each beam of light may be directed to a different detection system, which may include one or more detectors. For example, one of the four beams may be directed into detection system 26. Detection system 26 may be configured to detect light scattered by the microspheres.

The scattered light detected by detection system 16 and/or detection system 26 is generally proportional to the volume of particles illuminated by the light source. Thus, the output signal of the detection system 16 and/or the output signal of the detection system 26 may be used to determine the diameter of a particle located in the illumination area or detection window. Furthermore, the output signals of detection system 16 and/or detection system 26 may be used to identify more than one particle that is stuck together or passes through the illuminated area at substantially the same time. Thus, these particles can be distinguished from other sample microspheres as well as calibration microspheres. Moreover, the output signals of detection system 16 and/or detection system 26 may be used to distinguish between sample microspheres and calibration microspheres based on size of dimensions as described herein.

The other 3 beams may be directed to detection systems 28, 30, and 32. Detection systems 28, 30, and 32 may be configured to detect fluorescent light emitted by the microspheres. Each detection system may be configured to detect fluorescence at a different wavelength or range of wavelengths. For example, one of the detection systems may be configured to detect green fluorescence. Another detection system may be configured to detect yellow-orange fluorescence, while the other detection system is configured to detect red fluorescence.

In certain embodiments, filters 34, 36, and 38 may be coupled to detection systems 28, 30, and 32, respectively. The optical filter may be configured to block fluorescence at wavelengths other than the wavelength that the detection system is configured to detect. Furthermore, one or more lenses (not shown) may be optically coupled to the respective detection systems. The lenses may be configured to focus scattered light or emitted fluorescent light onto a photosensitive surface of the detector.

The output current of the detector is proportional to the fluorescent light impinging on it and generates a current pulse. The current pulses may be converted to voltage pulses, low pass filtered, and then digitized by an a/D converter. A processor 40, such as a DSP, integrates the area under the pulse to provide a number representing the magnitude of the fluorescence. Further, the processor may perform additional functions described herein (e.g., monitoring one or more parameters of the flow cytometer type measurement system, changing one or more parameters in real time based on the monitored parameters, etc.). As shown in fig. 1, processor 40 may be coupled to detector 26 via a transmission medium 42. Processor 40 may also be indirectly coupled to detector 26 via a transmission medium 42 and one or more other components, such as an a/D converter. The processor may be coupled to other detectors of the system in a similar manner.

In some embodiments, the output signal generated by the fluorescence emitted by the microsphere may be used to determine the identity (identity) of the microsphere and information about the reaction occurring at the surface of the microsphere. For example, the output signals of two of the detection systems may be used to determine the identity of the microsphere, while the output signals of the other detection system may be used to determine the reaction occurring on the surface of the microsphere. Thus, the choice of detector and filter may vary depending on the type of dye bound or bound to the microsphere and/or reaction being measured (i.e., the dye bound or bound to the reactant involved in the reaction).

The detection systems (e.g., detection systems 28 and 30) used to determine the identity of the sample microspheres may be APDs, PMTs, or other light detectors. As described herein, changes in gain as a function of temperature as described herein may be corrected for APDs in real time. The detection system (e.g., detection system 32) used to identify the reaction occurring at the surface of the microsphere may be a PMT, APD, or other form of photodetector. The PMT may be corrected by using a simple multiplier derived from the PMT characteristic curve, which may be applied to the output signal of the PMT as described herein. The probe and measurement system may also be configured as described herein.

Although the system shown in fig. 1 includes two detection systems having two different detection windows for distinguishing between microspheres having different staining characteristics, it should be understood that the system may include more than two such detection windows (e.g., 3 detection windows, 4 detection windows, etc.). In such embodiments, the system may include additional beamsplitters and additional detection systems with other detection windows. Furthermore, filters and/or lenses may be connected to each of said additional detection systems.

In another embodiment, the system may include two or more detection systems configured to distinguish different materials reacting on the surface of the microsphere. The different reactant materials may have different dyeing characteristics than the microspheres.

Examples of other measurement systems that can be used to perform the methods described herein are set forth in U.S. Pat. No. 5,981,180 to Chandler et al, U.S. Pat. No. 6,046,807 to Chandler et al, U.S. Pat. No. 6,139,800 to Chandler et al, U.S. Pat. No. 6,366,354 to Chandler et al, U.S. Pat. No. 6,411,904 to Chandler et al, U.S. Pat. No. 6,449,562 to Chandler et al, and U.S. Pat. No. 6,524,793 to Chandler et al, all of which are fully incorporated herein by reference. The measurement system described herein may also be configured in the manner described in these patents.

In flow cytometer type measurement systems, scattered light and bead identity detection are typically performed using Avalanche Photodiodes (APDs) as optical sensors. An advantage of APDs over other detectors is that by applying a reverse bias voltage, the output current level or "gain" of the APD can be varied over a wide range. The gain, which may be expressed in terms of electrons caused to flow by a constant number of incident photons, is proportional to the magnitude of the applied bias voltage. Unfortunately, the transition from incident photons to output electrons is strongly temperature dependent. Thus, APD is quite temperature dependent, to a greater extent than any other element in a flow cytometer type measurement system.

Accordingly, one embodiment of a method of controlling one or more parameters of a flow cytometer type measurement system includes monitoring a temperature proximate to the flow cytometer type measurement system. The method also includes changing a bias voltage of an APD of the measurement system in response to the temperature.

Each APD is rated by the manufacturer for a reverse bias voltage (V60) at which an output current that is 60 times greater than a silicon diode under approximately the same illumination can be obtained. The value of V60 ranges from tens of volts to over 100 volts depending on the individual device.

Since the output of an APD is non-linear with respect to temperature, a constant compensation factor cannot be used over the entire operating range of the APD. Empirical measurements of current output versus temperature can be used to develop a comprehensive compensation method. In other words, empirical data can be employed to determine a correction factor that is used to change a parameter of the APD. In particular, empirical data can be employed to vary the bias voltage of the APD to substantially correct for variations in gain of the avalanche photodiode due to temperature.

To characterize the response of an APD with empirical data, an approximately constant light level is applied to the APD at one or more temperatures. The current output of an APD at multiple bias voltages is recorded at one or more given temperatures. The temperature is changed (e.g., increased by an integer number of degrees) and then the measurements at multiple bias voltages are repeated again. The resulting data set (e.g., as shown in fig. 2) adequately describes the illumination-current versus temperature curve for a particular V60 device. These measurements can be repeated for APDs with different V60 ratings in order to obtain the response of a number of different devices.

In one embodiment, the bias curve table may be used to correct the temperature in the following manner. Upon initial system calibration, calibration microspheres that emit light of known intensity are introduced into the system. The calibration microsphere flows through the system and, as the calibration microsphere is measured by the measurement system, the bias voltage is varied until a predetermined signal level is obtained from the APD. The current reading of the APD is then inserted into the table (R-value) using the detector's V60, bias voltage at the predetermined signal level, and temperature as an index into the APD response table.

In another embodiment, the bias curve table may be generated in the following manner. The light-sensitive area of the APD can be remotely illuminated via an optical cable using a constant light output source such as a Light Emitting Diode (LED). The APD may then be placed in an environmental chamber where the ambient temperature to which the APD is exposed may be varied. The measurement system then records the APD current output (R-value) as the temperature and bias voltage applied to the APD change.

During normal sample operation, the temperature closest to the flow cytometer type measurement system may be monitored. The desired relative current, temperature, and empirical data may then be used to determine the bias voltage. For example, the R value, measured temperature, and V60 parameter can be used as inputs to an APD response table to find the corresponding bias voltage. If the measured temperature is between the entries of the table, the reading corresponding to the closest temperature entry is interpolated to obtain the optimal bias voltage. The bias voltage obtained from the table is applied to the APD to correct for its gain variation with temperature. Since the time of the sample run is typically within two minutes, and the temperature does not vary much during this time, it is generally sufficient to make a single offset correction at the beginning of the sample run and maintain the offset during the run. In other words, the bias voltage may be changed before the measurement system makes a sample measurement, while the bias voltage may be substantially constant during the sample measurement. However, it is possible that the temperature closest to the measurement system is monitored over time as the sample runs, and the bias voltage of the APD can be changed accordingly. In this manner, monitoring of temperature and changes to the bias voltage of the APD are performed in real time.

The indicator channel of some flow cytometer type measurement systems includes a photomultiplier tube (PMT) that serves as a light sensitive detector. The indicator pathway may generally be defined as a pathway for identifying materials involved in a reaction occurring at the surface of the microsphere or materials bound to the surface of the microsphere. The PMT generates a current proportional to the amount of light that illuminates the photocathode, the bias voltage applied, and the number of internal dynodes (internal dynodes) within the PMT. In a flow cytometer, the bias voltage of the PMT is typically used as the "control" point to normalize the current output for a given level of fluorescence. The methods currently employed to obtain the normalized voltage during calibration are empirical, measurements are taken, and then an educated guess is made for the PMT bias setting that is likely to produce an output closer to the desired value. Typically, multiple iterations are required before the output error level reaches an acceptable range. Therefore, it is advantageous to shorten the calibration time, thereby reducing the amount of calibration reagents (reagent) used to find the optimal PMT voltage. Several different methods that can make the calibration process faster than the present case will be described below.

Because of the substantially linear response to temperature, it is much simpler for a PMT to compensate for temperature variations than an APD. For example, one embodiment of a method for controlling one or more parameters of a flow cytometer type measurement system includes monitoring a temperature proximate to the flow cytometer type measurement system. The temperature is typically measured as close as possible to the PMT, although the precise location is not critical since the rate of change of the PMT temperature is relatively slight. The method also includes employing a characteristic curve of the PMT to change an output signal of the PMT of the measurement system in response to temperature to substantially correct for changes in an output signal gain of the PMT due to temperature. The gain of the PMT changes nearly linearly in response to temperature. In addition, the characteristic curve of the PMT varies depending on the detection wavelength and the cathode structure. In this manner, the response of the PMT to temperature at a given detection wavelength and cathode configuration can be represented by a simple linear relationship, as shown in FIG. 3, from Photomultiplier tube theory to Application (Photomultiplier tube to Principal to Application) of Hamamatsu Photonics K.K1994, which is hereby fully incorporated by reference.

As previously mentioned, since the variation of the gain of a PMT with temperature is much smaller than that of an APD, it is generally not necessary to compensate the device by changing the gain or determining the bias voltage. Instead, it is sufficient to use a simple multiplier derived from a PMT characteristic curve, such as the curve shown in fig. 3, which can be applied to the final PMT reading by the reporting software.

To calibrate the PMT, calibration microspheres with known amounts of fluorescence are provided to the instrument and flowed through the system as if normal samples were taken. When the measurement system takes measurements of the calibration microspheres, the bias voltage is varied until a predetermined signal level is obtained.

The method is an iterative process in which statistics for a set of microsphere readings are calculated and the process is terminated when a desired tolerance is obtained. If the error is not small enough, the results of the first two iterations may be used to predict the next PMT bias setting. In this process, a linear equation is used: y-m x + b, where the slope m is defined by the previous bias and the resulting fluorescence measurement. If the bias voltage versus current gain transfer function of the PMT is linear, the final result can be obtained directly and tested by another measurement. However, since the voltage-current gain transfer function of the PMT grows exponentially with increasing bias voltage, the linear approach is only effective over a relatively small segment of the curve, and therefore several iterations are required to meet the final tolerance requirements.

Interestingly, when the voltage-gain relationship of the PMT is plotted on a log-log graph (see fig. 4), its transfer function appears as a straight line. The data in FIG. 4 is from photomultiplier-theory to applications, K.1994, Hamamatsu Photonics K.K.

As described earlier, the number of internal dynodes and the applied bias voltage determine the current amplification of the PMT. For a fixed light level, the output current is proportional to V to the nth power, as shown in equation 1, where V is the applied bias voltage, N is the number of internal dynodes, and a is a proportionality constant that encompasses several physical properties of the PMT.

i＝A*V^N (1)

Taking the logarithm of both edges of equation 1 yields the following equation:

log(i)＝N*log(V)+log(A) (2)

this equation can be written as a simple and common first order linear equation:

y＝m*x+b (3)

where y is log (i), m is N, x is log (v), and b is log (a). With logarithmic conversion it is now possible to perform a shortened calibration operation using only three sample measurements.

For example, in one embodiment, a method for controlling one or more parameters of a flow cytometer type measurement system includes setting a voltage of a PMT of the measurement system to a first value and a second value. The method also includes measuring an output current of the PMT at the first value and the second value. In addition, the method includes determining a calibration voltage of the PMT from a logarithm of the first value and the second value versus a logarithm of the output current at the first value and the second value. The method also includes applying a calibration voltage to the PMT and testing the PMT to determine whether one or more parameters of the PMT are within a predetermined tolerance.

One specific example of such a method is summarized by steps 1-7 below:

1. the PMT voltage is set to a value that is closest to or at the low end of its range (V ═ V)_L) And obtaining a measurement (i ═ i)_L)。

2. The PMT voltage is set to a value that is closest to or at the high end of its range (V ═ V)_H) And obtaining a measurement (i ═ i)_H)。

3. The logarithm is taken for all four values.

4. The slope m and intercept b are calculated.

5. Solving for the target PMT setting (in logarithmic space) x_cal。

6. Get x_calTo obtain PMT calibration voltage V_cal。

7. Application of V_calAnd tested to determine if the desired tolerance is met.

The method has been verified to converge within tolerance successfully each time. If the tolerance is not met, V can be obtained by using the previous calculation_cal，i_calAnd V_H，i_HNew slopes and intercepts are generated in log space, which may yield acceptable results. Point V_cal，i_calWhich may be relatively close to the final PMT voltage, only a short traversal (traversal) of the smoke new line is required to produce acceptable results. In this case, four sample measurements will be used to find the appropriate calibration voltage.

Another method for calibrating a probe of a flow cytometer type measurement system advantageously reduces the number of calibration iterations using successive approximation. In one embodiment, a method for controlling one or more parameters of a flow cytometer type measurement system includes determining a calibration voltage for a probe of the measurement system using a successive approximation method, as shown in step 50 of FIG. 5. When all calibration voltages have been applied to the probe but a successful calibration has not been achieved, the method will exit the calibration with a failure, as shown in step 52. Since the detector may be an APD, PMT, or any other detector suitable for use in the measurement system, each detector voltage may be compared to a detector voltage limit, as shown in step 54. If the calibration voltage exceeds the voltage limit, a different calibration voltage may be determined by repeating at least step 50.

As shown in steps 56, 58, and 60, the method applies a calibration voltage to the probe, collects data from the probe, and may include constructing a histogram of the collected data, calculating a peak value of the histogram, and comparing the histogram peak value to a calibration target peak value. If the histogram peak is close enough to the calibration target peak, the calibration may terminate, as shown in step 62.

The method may also include determining whether the histogram peak is above the calibration target peak, as shown in step 64. The output of step 64 may be used to correct the next calibration voltage generated by the successive approximation in step 50.

Although the method is described above with respect to a histogram, it should be understood that any suitable statistical measure may be employed to perform the method. For example, any suitable method of determining the level of the detector signal may be employed, which may (but need not) include determining statistical methods of measurement, such as averages, medians, etc., from a collection of bead samples.

In particular, successive approximation attempts to make the measured value equal to the target value by setting and clearing bits in the command word only up to N times. In one embodiment, the method may include collecting and processing detector samples to determine a detector signal level. In another such embodiment, the method may include comparing the detector signal level to a calibration target signal level, and if the detector signal level is higher than the calibration target signal level, decreasing the detector bias voltage and repeating the determination of the calibration voltage. In yet another such embodiment, the method may include comparing the detector signal level to a calibration target signal level, and if the detector signal level is not within a predetermined range of the calibration target signal level, repeating the determination of the calibration voltage until all desired detector voltage levels have been tried.

One specific example of such a method includes the steps of:

1. initializing bit mask (bit mask) and DacCmd values to 2^N. For a 12-bit Dac ("digital-to-analog converter"), N — 12. In this example, the bit mask is 4096 and the DacCmd value is 4096. The Dac may include any suitable Dac, such as Dac, available from Analog Devices, Inc.

2. The current mask bit is used to clear the corresponding bit in DacCmd. We drive either beyond the target or beyond the detector maximum voltage limit.

3. The mask is shifted to the right by one bit (e.g., to the next most significant bit).

4. If the mask is 0, all possible bits have been detected without having obtained sufficient alignment. The method may proceed to step 12.

5. The mask and DacCmd are summed to set the next most significant bit.

6. A detector voltage corresponding to the DacCmd binary value is determined. The detector voltage is compared to a detector breakdown voltage or maximum voltage. If the voltage exceeds the detector breakdown voltage, step 2 is returned to.

7. The DacCmd value (e.g., the voltage) is sent to the measurement system.

8. Waiting for the voltage change to take effect.

9. The new histogram peak is compared to the calibration target peak for that channel. If the histogram peak is greater than the calibration target, return to step 2.

10. If the histogram peak is not sufficiently close to the desired target, return to step 3.

11. And (4) passing the calibration. The method ends.

12. The calibration fails. The method ends.

The exemplary method described in steps 1-12 may include any of the other steps described herein.

Some flow cytometer type measurement systems use hydrostatic focusing techniques to separate beads as they flow through two detection windows for individual measurement. The measurement windows have a fixed size and physical separation. For example, the distance between the points of illumination of the light source in the measurement system defines the separation.

Changes in the velocity of the underlying liquid transporter will change the length of time the bead is within the detection window, as well as the length of time that the bead is spaced from one window to the next. The final reading is proportional to the length of time the beads are present within each detection window. In addition, the system uses the intra-window transit time to determine when the second detection window is active (i.e., when the bead is located in the second detection window to be measured). If the alignment in time of the sample measurement and the actual bead presence differs from the values obtained during calibration, or the duration (dwell) time in the illumination window differs, the accuracy of the measurement will be degraded.

The effect of temperature on the velocity change of the liquid is most influential if the measurement system is configured to maintain the pressure of the liquid substantially constant during the measurement of the sample microspheres. Viscosity is defined as a measure of the flow resistance of a liquid. The volume of liquid flowing through a pipe of diameter R and length L at pressure P per unit time can be expressed by the Poiseuille (Poiseuille) equation:

V/T＝(π*R⁴*P)/(8*N*L) (4)

where V/T is the volume per unit time (proportional to speed) and N is the viscosity in poise (poise). The capillaries of the flow capsule, although rectangular in dimension rather than circular, can be considered as simple tubes. Therefore, as defined in the Poiseuille equation, the bead velocity is inversely proportional to the viscosity of the liquid carrier.

The main component of the liquid used as the bead transporter of a flow cytometer type measurement system is water. The viscosity varied from 1.139 centipoise to 0.7975 centipoise with a significant change of 43% over the operating temperature range of 15 ℃ to 30 ℃. The Viscosity values above are obtained from The "Viscosity of Water between 0 and 100 ℃ in The Handbook of Physics & Physics" of The 61 st edition (The viscocity of Water 0 to 100 ℃). The sheath (sheath) and sample liquid velocities also varied by about 43% as did the beads. Thus, the operating temperature may be measured and then used to determine the viscosity of the liquid. Accordingly, the velocity of the liquid can be determined from a table, a Poiseuille equation, or a predetermined value of velocity versus temperature. In such embodiments, the method may include controlling the pressure of the liquid during the measurement of the sample microspheres based on the velocity.

In addition, the velocity of the liquid can be used to determine the bead velocity. Also, the transfer time can be extracted and corrected in real time. If the temperature of the liquid does not substantially change during the sample measurement, the monitoring of the temperature and the determination of the velocity may be performed prior to the measurement of the sample microspheres by the measurement system. However, these steps of the method may also be performed in real time.

Accordingly, one method for controlling one or more parameters of a flow cytometer type measurement system includes monitoring a temperature of a liquid to be flowed through the flow cytometer type measurement system. Sample microspheres are placed in the liquid. The method also includes determining a velocity of sample microspheres in the measurement system based on the viscosity of the liquid at the temperature. In some embodiments, the method further comprises determining a length of time that one of the sample microspheres will be present in a detection window of the measurement system based on the velocity. In another embodiment, the method includes determining a length of time that one of the sample microspheres moves from one detection window to another detection window of the measurement system based on the velocity. Further, the method may include determining when one of the sample microspheres will be present in a detection window of the measurement system based on the velocity. Furthermore, the method may comprise controlling the sampling interval of one or several detection windows of the measurement system, thereby compensating for the velocity.

The transfer time within the window may be measured and saved in a non-volatile memory of the system or in a computer that controls the system during an initial calibration. The measured transit time may then be used in subsequent sample runs to properly time the sampling interval of the second detection window. The transfer time within the window can be shortened or lengthened to compensate for viscosity changes. The temperature at which the system is calibrated versus the current temperature may be used to determine the amount of correction to be applied. The temperature versus viscosity factor profile may be stored in a computer controlling the system or in a non-volatile memory of the system. In either case, the transit time correction factor may be calculated from the table and applied before the sample run begins. Alternatively, any other suitable method known in the art may be used to determine the correction factor.

The method may further comprise determining one or more characteristics of an output signal of the measurement system from the velocity. For example, the length of time the beads are present in the detection window determines the amplitude and shape of the detector's output electrical pulse. The pulses then pass through an analog low pass filter which has the significant effect of reducing the amplitude and broadening the pulse width. The filtered pulse is digitized and the area under the pulse is calculated to yield a value approximately proportional to the light level.

Further, the method may include correcting an error in the output signal due to velocity using the correction factor. The correction factor may be determined using empirical measurements. It is apparent that empirical measurements are used to construct a table of correction factors for pulse width variations due to flow rate variations. This table may be stored in the memory of the system or in a control computer connected to the system.

Another method of compensating for speed changes due to temperature changes is to vary the applied liquid pressure proportionally to changes in viscosity. This will cause the velocity to remain constant so there is no significant change in time within or between each measurement window. The method may be performed in real time or in real time at the start of a sample run, or via a predetermined table calculated from the poiseuille equation, or by other methods, to dynamically set the appropriate pressure.

These methods have proven to provide substantial improvements over constant pressure schemes, however, other compensation for temperature variations may be desirable. Therefore, another approach is described herein that can be used separately from or in conjunction with the above approach to provide a fine-tuning mechanism. Unlike the above-described method, this method employs an optical mechanism. Further, the method may use measurement and control algorithms. However, as described herein, this approach is relatively inexpensive and fast, apart from the additional optical mechanisms and algorithms.

When the optical elements of the flow cytometer type measurement system are assembled, the distance between the illumination points (e.g., laser points) is initially set. As the distance between illumination points (e.g., or beams) decreases, the effect of velocity variations on the bead transit time is minimized due to the shorter distance the bead travels between detection windows.

The minimum separation distance is further defined by the vertical illumination profile (i.e., the profile of each beam in a direction generally parallel to the direction of microsphere flow through the measurement system) of each beam. For example, if the beam intensity decreases rapidly from peak to shoulder, and there is no second maximum, it is possible to place the beams relatively close to each other because light from one source does not spill over into other illumination points. Care needs to be taken to avoid overlapping beams as such overlapping can result in complex compensation schemes between the sorting and indicator channels, resulting in reduced sensitivity.

As previously mentioned, it is important to keep the bead transit time between illumination points substantially constant, which in turn substantially fixes the velocity and the time the microspheres spend within the respective illumination window.

One method for maintaining the bead transit time substantially constant involves measuring the average time a bead passes through two detection windows in real time and controlling the applied pressure as needed to keep the transit time constant. According to one embodiment, a method for controlling one or more parameters of a flow cytometer type measurement system includes measuring a time for a microsphere to move from a first detection window of the flow cytometer type measurement system to a second detection window of the measurement system. In one embodiment, the time may be an average time. The microspheres may be sample microspheres or calibration microspheres. The measuring of time may comprise measuring light scattered by the microspheres in the first and second detection windows. In another embodiment, the measuring of time may comprise measuring light scattered by the microspheres in the first and second detection windows with one detector. In one such embodiment, light scattered by the microsphere in the first and second detection windows is directed by a beam splitter to a detector. The method further comprises varying the applied pressure of the measurement system such that the time is substantially constant. The method may be operated in real time. Embodiments as described above may include any other steps described herein.

Unfortunately, the current optical design of most flow cytometer type measurement systems makes it impossible to detect every bead that passes through the second detection window where typically only the indicator fluorescence is measured, because the fluorescence emission (which is not known a priori) is not constant for every bead, and is very likely to be zero for some beads. The obvious solution is to add another optical detector to measure the light of the second illumination source scattered by the beads, but this adds significant cost to the system, since additional electronic and digital processing chains are required to process the new signal.

The proposed method is simple and inexpensive, since it involves measuring the scatter in two detection windows using the same scatter detector. Since the current optical layout prevents scattered light in the second (indicator) window from reaching the scatter detector, the detector must be repositioned so that it receives all light emitted or reflected by the beads. After this step is completed, distinct peaks roughly proportional to the scatter from each light source can be separately identified by downstream electronics.

FIG. 6 depicts one embodiment of a measurement system that may be used to perform the methods described herein. As shown in fig. 6, the measurement system includes light sources 70 and 72. The light source 70 may be, for example, a laser emitting light at a wavelength of about 639 nm. The laser may be adapted to provide illumination for a sorting channel of the measurement system. The light source 72 may be, for example, a laser emitting light having a wavelength of approximately 532 nm. The laser may be adapted to provide illumination for an indicator channel of a measurement system. Note that the illuminated area of each laser does not coincide with the axis of bead flow (not shown). Other light sources may be used in the above examples. For example, the light source and its wavelength may vary depending on the sample to be measured.

As shown in FIG. 6, both light sources 70 and 72 illuminate cuvette 74. In particular, light sources 70 and 72 are configured to illuminate beads 76 as they flow through cuvette 74. As further shown in fig. 6, light sources 70 and 72 may be configured to illuminate the beads at substantially opposite angles. However, it should be understood that the light source may illuminate the beads at any suitable angle.

Light scattered by the beads due to illumination by the two light sources may be collected by lens 78. Lens 78 may comprise any suitable lens known in the art. Further, the lens 78 may be replaced by a reflective collector (collector) or may not be included in the system. Although the lens 78 is shown as collecting light at a collection angle of about 90 (relative to the light sources 70 and 72), it should be understood that the lens may be disposed at any suitable collection angle relative to the light sources.

The light collected by the lens 78 is directed to a beam splitter 80. The beam splitter 80 may include any suitable optical component known in the art, such as a glass plate or a spectral filter. The beam splitter 80 is configured to direct a portion of the light collected by the lens to a detector 82. The detector 82 may be configured to detect light scattered by the beads due to illumination by the two (or more) light sources. In this manner, detector 82 may be configured to detect light scattered by the beads at wavelengths of approximately 532nm and 639nm, as opposed to the example light source provided above. The detector may comprise any suitable detector known in the art, such as a CCD device.

The detector 82 will therefore detect two different scatter signals of a single bead. These scattered signals are detected at different wavelengths, which are determined based on the wavelength of the light source. Since each light source illuminates the bead at a different time as it passes through the cuvette, the time at which the different scattered signals are detected can be used to measure the time at which the bead or microsphere moves from the first detection window of the measurement system to the second detection window of the measurement system.

Further, the beam splitter 80 is configured to transmit other portions of the light collected by the lens. The portion of the light transmitted may be directed by the optical assembly 84 to a classification section 86 of the detection subsystem of the system. The optical assembly 84 may include, for example, a folding mirror, a dichroic filter, a partially transmissive mirror, or other suitable assembly known in the art. Alternatively, the optical assembly 84 may not be included in the system, depending on factors such as the arrangement of the classification components of the detection subsystem. The classification portion of the detection subsystem may include any component known in the art. In some embodiments, the classification portion of the detection subsystem may be configured as described in FIG. 1. Another portion of the light transmitted by beamsplitter 80 may be directed to an indicator channel (not shown) of the detection subsystem. Although the system uses a first illumination region for classification and a second region for indicator signals, the manner of use in a device employing this technique is not limited to these embodiments. Fluorescent or scattered light may be used for another purpose, for example, to measure fluorescent indicators or other dyes in cells, beads, and other particles.

The fluorescent emission (if any) directed by the beamsplitter 80 to the detector 82 will be added to the scattered signal, but its effect is not so important because its amplitude is much smaller than that of the scattered light. As described above, the implementation shown in FIG. 6 employs a beam splitter 80, which may be a wavelength splitter, that redirects scattered light to a repositioned detector without changing the spectrum applied to the classification detectors. Obviously, other embodiments are possible. For example, it is conceivable that the detector may be arranged so as not to contain additional components. The system shown in fig. 6 may be further configured as described herein.

Another embodiment of a method for controlling one or more parameters of a flow cytometer type measurement system includes measuring an average time for a microsphere to move from a first detection window of the flow cytometer type measurement system to a second detection window of the measurement system. The microspheres may include sample microspheres, calibration microspheres, or both. The method further includes comparing the average time to a reference time, the reference time being the time during which the reference microsphere moves from the first detection window to the second detection window. The method may or may not include the measurement of the reference time. Further, the method includes changing the applied pressure of the measurement system if the difference between the average time and the reference time is greater than a predetermined value. In some embodiments, the predetermined value may be selected to compensate for known time varying mechanisms of the measurement system. In one embodiment, varying the applied pressure comprises increasing the applied pressure if the average time is greater than the reference time. In a different embodiment, varying the applied pressure includes decreasing the applied pressure if the average time is less than the reference time. The method can also be carried out in real time.

The above-described method provides a technique for directly controlling the system pressure such that the time between successive scatter pulses is approximately constant. The techniques may be implemented using electronic hardware (e.g., counters, digital comparators, etc.) or software using sampled signals measured by a digital signal processor or other suitable processor. In either embodiment, the method is simulated, with the same results. A high level description of the algorithm is provided below in steps 1-6, and an example of a burst is shown in fig. 7.

1. When the system is calibrated at known pressures and temperatures, the average transit time between successive scatter pulse peaks is measured and stored for later reference.

2. During normal sample acquisition, the first scatter pulse from the red laser (or any other light source that first illuminates the beads) starts a timer. For example, as shown in FIG. 7, at t₁At the instant, the scattered pulse corresponding to the laser illumination with wavelength 639nm is detected. Thus, the timer starts at t₁。

3. When the second scatter pulse arrives, the timer is stopped. For example, as shown in FIG. 7, when a scattered pulse corresponding to laser irradiation with a wavelength of 532nm is at t₂When the time is detected, the timer is stopped.

4. The value of the timer is then used to compare with the transit time measured during the calibration operation.

5. If the timer value is significantly higher than the calibration time, one or more parameters of the pressure source (e.g., pump) may be changed to increase its pressure. The parameters of the pressure source may be changed by the processor. Or, if t₂And t₁Is greater than t_calThe pressure of the pressure source may be increased. t is t_calMay be a predetermined value defining an acceptable amount of change in the transit time of the beads.

6. If the timer value is significantly below the calibration time, one or more parameters of the pressure source may be changed to decrease its pressure. The one or more parameters may be changed by the processor. Or, if t₂And t₁Is less than t_calThe pressure of the pressure source may be reduced. T used in step 5 and step 6_calHave the same value.

In order to keep the "control system" relatively stable, several situations may be considered. For example, the method may be performed such that the system is not controlled to try or make a forward or reverse pressure correction for each bead event passing through the system. Some averaging method may be employed to compensate for a known time-varying mechanism called "bead jitter", which is believed to be caused, at least in part, by the velocity gradient of the sample core. Also, the threshold value for the time error causing the pressure correction should be carefully selected. The magnitude of the error is preferably used as an input to a controller that determines the amount of pressure correction. It is likely that a classical integral-differential controller can be used for good operation.

While the above-listed correction factors may be used to correct a substantial portion of the measurement error prior to measurement of the sample microspheres, fine corrections may also be made during the measurement to compensate for residual errors that may exist after the above-described techniques are applied. For example, one method for controlling one or more parameters of a flow cytometer type measurement system includes monitoring one or more parameters of the measurement system as the measurement system takes measurements of sample microspheres. The method also includes changing one or more parameters in real time based on the monitored parameters. For example, as described above, the one or more parameters that are monitored and changed may include parameters of a PMT of the measurement system. In addition, error sources not identified in this specification can be eliminated using this process.

Flow cytometer type measurement systems identify microspheres that pass through the system based on the measured intensity (intensity) of two or more dyes within the microspheres. This identification technique may also be used to identify calibration microspheres containing a known amount of fluorescence intensity in all channels (both the indicator channel and the classification channel). After the measurement results of the calibration microspheres are known, fine correction factors may be applied to the indicator and/or the sorting channel for sample microsphere measurements.

This technique may be complicated when distinguishing between calibration microspheres and sample microspheres. For example, a new spectral address for a calibration microsphere may be created based on a combination of dye levels, but this may reduce the multiplexing capability of the system by N-1. Another technique identifies calibration microspheres by making their diameter larger or smaller than the diameter of the sample microspheres.

The measurement system may measure light scattered by the microspheres at a 90 angle to the plane of illumination. The level of scattered light is used to identify a plurality of microspheres that may stick together or may pass through the illuminated area substantially simultaneously. For example, scattered light is generally proportional to the volume of all particles present within the illuminated area; thus, the scattering signal is stronger for multiple microspheres than for a single microsphere. Since most microspheres typically pass through the illuminated area as a single object, by considering population events, those events that do not belong to a single bead are easily identified. Typically, two, and sometimes three, microspheres come together and produce a signal that is stronger than the signal produced by a single microsphere. The level of the scattered signal from a single microsphere is typically carried out during the progress of the assay, as the assay format has an effect on the scattered signal.

Calibration microspheres having a diameter less than, rather than greater than, the diameter of the sample microsphere should preferably be used because the calibration microspheres can be more easily distinguished from any number of microsphere combinations passing through the illumination zone. Thus, parameter monitoring of the measurement apparatus may be performed using measurement data of calibration microspheres having a diameter smaller than the diameter of the sample microspheres. In addition, the one or more parameters that are monitored and changed may include output signals generated by a detector of the measurement system that are responsive to light scattered by the sample microspheres. For example, if the ratio of the calibration microsphere diameter to the sample microsphere diameter is known, it is also possible to use the calibration microsphere to fine tune the scattering measurements.

At least some of the calibration microspheres may also have different spectral addresses. Thus, a series of different calibration microspheres may be used to enhance the calibration method described above. For example, by using the diameter as a first discrimination point, the spectral address of the calibration microsphere can be used as a second discrimination point in the calibration space, just as in the sample space. Having multiple calibration levels that are sufficiently separated within the classification space to discriminate the identity of the microspheres may be used in implementations below.

For example, one or more parameters that may be monitored and changed may include linearity in the measurement of sample microspheres. The measurements that monitor or change the parameters of the measurement device may include measurements of the classification channels of the measurement system. In this embodiment, the change to the parameter of the measurement device preferably corrects for any non-linearity in the measurement. As such, multiple correction levels may be used to detect and correct for non-linearities in the hierarchical space. Current measurement systems use only a single point calibration and therefore errors due to system non-linearity cannot be corrected. In a two-dimensional representation of a two-dye bead system, this non-linearity can be treated as a variation of the classification space in the plane based on the observed position of the classification microsphere. Correcting for non-linearity improves the microsphere classification accuracy in this plane. The technique can be extended to any dimension with similar effect.

Multiple calibration levels may also be used to detect and correct for non-linearities in the indicator signal. Similar to the techniques described above, the indicator channel may also be subjected to a single calibration point in current measurement systems. Detecting and correcting for non-linearities in the indicator pathway may operate as described above. For example, measurements made during the time that a parameter of the measurement system is being monitored and changed may include measurements of an indicator channel of the measurement system. Furthermore, measurements made during the monitoring and changing of parameters of the measurement system may include measurements of indicator channels and classification channels of the measurement system. In this manner, non-linearities in the classification and indicator channels may be monitored and corrected for substantially simultaneously.

In another example, the one or more parameters of the measurement system that may be monitored and changed include the dynamic range of the measurement system. For example, multiple calibration levels may also be used to make real-time determinations of the dynamic range of the system. The measurement system has a limited linear range. By using different indicator calibration levels on one or more uniquely identified calibration microspheres, it is possible to identify lower and/or upper limits of detection by the detector at which the system becomes non-linear due to signal clipping.

In certain embodiments, multiple calibration levels may be used to determine the system health status classification. As such, the one or more parameters that are monitored and changed may include measuring system health. Measuring the system health may include classifying the health of the channels, indicating the health of the channels, or both. For example, if the above-described set of methods cannot compensate the system for temperature or other effects, the calibration microsphere fluorescence classification level will be higher than it would be expected. A threshold level may be set and the calibration microsphere fluorescence classification level may be compared to the threshold. If the calibration microsphere fluorescence classification level falls on a selected side of the threshold level, a warning may be provided to the system operator or sent to a computer coupled to the measurement system informing that the measurement result is suspect. The warning may be a visual output signal and/or an audio output signal. In a similar manner, multiple calibration levels may be used to determine the indicator system health status. Similar to the determination of the health status of the classification system, uncorrectable errors in the indicator system may be identified and reported to a system operator or a computer connected to the measurement system.

Also, multiple calibration levels may be used to extend the linear dynamic range of the indicator channel. As such, changing a parameter of the measurement system may include extending a linear dynamic range of an indicator channel of the measurement system. By including calibration microspheres of several brightness levels present in the non-linear region, it is possible to map the actual measured fluorescence level onto its linear equivalent. By interpolating between the individual calibration microsphere values, a smooth mapping from the measured curve to the desired curve can be constructed from the calibration data. Thus, if the curve is used to adjust sample microspheres in a non-linear region, the linear, usable measurement range of the system can be significantly extended.

In the above description, several measurement error factors (detectors) and real-time correction techniques for them, respectively, have been identified. In addition, a real-time fine tuning method using small diameter calibration microspheres that can be included in a microsphere sample mixture is created. Additional features of the fine tuning process include real-time identification of system health, correction for non-linearities in one or more channels, and significant extension of the available indicator dynamic range for the measurement system.

Program instructions to perform methods such as those described herein may be transmitted over or stored on a carrier medium. The carrier medium may be a transmission medium such as a wire, cable, or wireless transmission link, or a signal propagating along a wire, cable, or link. The carrier medium may be a storage medium such as read-only memory, random access memory, a magnetic or optical disk, or a magnetic tape.

In one embodiment, a processor may be configured to execute program instructions to carry out the computer-implemented method according to the above-described embodiments. The processor may take a variety of forms, including a special purpose processing board using a digital signal processing chip or field programmable gate array, a personal computer system, a mainframe computer system, a workstation, a network appliance, an internet appliance, a Personal Digital Assistant (PDA), a television system, or other device. In general, the term "computer system" may be broadly defined to include any device having one or more digital signal processing elements or other processing elements.

The program instructions may be implemented in a variety of ways including procedure-based techniques, component-based techniques, and/or object-oriented techniques, among others. For example, the program instructions may be implemented using ActiveX controls, C + + objects, JavaBeans, Microsoft Foundation Classes ("MFC"), or other techniques or methods, as desired. When an FPGA implementation is employed, a high-level language such as VHDL can be used to design the signal processing circuitry embedded within the device.

Those skilled in the art, having the benefit of this disclosure, will appreciate that: the present invention is believed to provide a method of controlling one or more parameters of a flow cytometer type measurement system. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention herein shown and described are to be taken as the presently preferred embodiments. Persons skilled in the art and technology to which this invention pertains will appreciate that elements and materials may be substituted for those illustrated and described herein, that parts and processes may be practiced, and that certain features of the invention may be practiced separately. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Claims (7)

1. A method for controlling one or more parameters of a flow cytometer type measurement system, comprising:

(a) setting a voltage of a photomultiplier tube of the measurement system to a first value and a second value;

(b) measuring the output current of the photomultiplier tube at the first value and the second value while the measurement system is measuring a set of calibration microspheres;

(c) determining a linear relationship from the log of the first and second values relative to the log of the output current at the first and second values and thereby determining a calibration voltage of the photomultiplier tube;

(d) applying the calibration voltage to the photomultiplier tube; and

(e) testing the photomultiplier tube to determine whether one or more parameters of the photomultiplier tube are within a predetermined tolerance by measuring the output current while the measurement system is measuring the set of calibration microspheres, wherein if the one or more parameters are determined to be outside the predetermined tolerance, the method further comprises:

(f) determining a new linear relationship in logarithmic space using the calibration voltage and calibration current and the output currents at the second and second values, and thereby determining a new calibration voltage; and

repeating the foregoing steps of (c) determining, (d) applying, (e) testing, (f) using, until the one or more parameters are determined to be within the predetermined tolerance.

2. The method of claim 1, wherein the step of determining a calibration voltage comprises determining a calibration voltage using successive approximation.

3. The method of claim 1, wherein said first value comprises a voltage value approaching a lower limit of said photomultiplier tube input voltage range.

4. The method of claim 1, wherein the second value comprises a voltage value approaching an upper limit of the photomultiplier tube input voltage range.

5. The method of claim 1, wherein the step of testing the photomultiplier tube comprises:

collecting data from the photomultiplier tube at the calibration voltage;

generating a histogram of the collected data;

calculating a peak value of the histogram; and

comparing the peak of the histogram to a target peak.

6. The method of claim 5, wherein the testing step determines that the one or more parameters of the photomultiplier tube are within the predetermined tolerance if the peak of the histogram is approximately equal to the target peak.

7. The method of claim 5, wherein the testing step determines that the one or more parameters of the photomultiplier tube are not within the predetermined tolerance if the peak of the histogram is not approximately equal to the target peak.