BACKGROUND LIGHT DETECTION SYSTEM FOR A FLOW

CYTOMETER

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of U.S. Provisional

Application Ser. No. 61/037,265, filed March 17, 2008, the entire contents of which are incorporated by reference.

TECHNICAL FIELD

[002] The present invention relates to a flow cytometry system and method for increasing the accuracy of measurement of the flow cytometry data. More particularly, the system and method has an array of processing elements that removes background light and nulls the background energy from the data stream prior to the analysis of the data.

BACKGROUND OF THE INVENTION

[003] Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus. A beam of light, usually laser light, of a single frequency (color) is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescent detectors. Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is possible to deduce various facts about the physical and chemical structure of each individual particle. FSC correlates with the cell size and SSC depends on the inner complexity

of the particle, such as shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness.

[004] Flow cytometry systems or flow cytometers convert light signals from a cell sample or particle to electronic pulses and then an analog-to-digital converter (ADC) converts the electronic pulses to channel numbers. The channel number represents flow cytometry data which can be presented on a display, such as a computer screen or printer, with the maximum acquired channel being presented at the end of the display object. As the technology for acquisition modules improves, cytometers are collecting wider dynamic data channel ranges. For example, instead of collecting four decades of dynamic data range, cytometers can now collect six decades of dynamic data range. This is significant when considering that a decade is a factor of 10 difference between two numbers measured on a logarithmic scale. For further example, four decades of information would result in approximately 10,000 data points. Whereas, the increased resolution and the resultant six decades would result in approximately 1,000,000 data points. Under this broader and larger data range, data previously viewed in channels for three or four decades of dynamic data range are now viewed in six decades of dynamic data range.

[005] Photo Multiplier Tubes (PMT) have long been the choice for photon detection in many instruments. Their ability to sense a wide range of photon concentration from a few photons to millions of photons without the loss of linearity is one of their strong characteristics. Their dynamic range can be in excess of six decades and makes them extremely useful in measurement systems where the dynamic range to be measured is large. By nature, PMTs are current output devices, where the output current is directly proportional to the number of photons impacting the photo cathode of the tube. All photon sources entering the photo cathode are contributors to the output current of the PMT. The output current is changed into a voltage using a Trans Impedance Amplifier (TIA). The TIA is normally an operational amplifier where the current from the PMT is fed into the summing junction with a feedback resistor from the output that provides the null current to the summing junction. The resultant voltage generated at the output of the TIA is proportional to the current entering the summing junction.

[006] One of the drawbacks to being able to make use of the total dynamic range of the PMT in pulsed light applications is the effect that background light in the optical system has on the TIA. The steady-state background light (DC light) entering the PMT produces an output current from the PMT as well as a corresponding output voltage from the TIA. When a pulse of light occurs, the pulse then appears on top of the steady state DC voltage caused by the background light. Because the total voltage output of the TIA is finite (usually in the range of 3 - 10 volts), the presence of the background light limits the total capability of the TIA output because of the DC background light occupying a significant portion of the max range of the amplifier, also known as the TIA voltage head.

[007] Another drawback of the background light is its impact on the system's

ADCs. A standard amplifier that is compatible with modern ADCs has a two volt maximum output and can have a theoretical dynamic range in excess of five decades. The interference caused by only a few hundred millivolts of background light can reduce the dynamic range of an ADC by up to a decade or more.

[008] In some systems, the interfering background light voltage was removed by coupling the TIA to the system amplifiers with a capacitor. The capacitor passes the output voltage created from the current output of the PMT output while blocking the DC background light component of the signal. In applications where the light pulses come in a random manner, this technique can produce a baseline drift that is proportional to the rate of pulses in the system and can generate errors in the measured output in excess of 10%. To allow for measurements of the pulses, this baseline drift is usually corrected using baseline restoration circuits. These highly tuned circuits form the basis of many instruments, and are difficult to design and implement.

[009] Another known method of treating the problems created by the background light exists in the telecommunications industry where the DC background light component of the signal on Avalanche Photo Diodes is removed using a low pass filter to detect the DC component and then feeding that signal back to the TIA to zero out the DC component of the signal. This method works well since the signals are constant in frequency and the low pass filter will not be affected by changes in frequency of the signal. And while this method is acceptable in the

telecommunications industry, when measuring randomly occurring pulses of light, this is not a practical solution for cytometry systems.

[0010] Another technique is to detect the DC baseline and try to restore the

DC component after the TIA. This technique removes the DC component from the amplifiers after the voltage leaves the TIA, but this method does not restore the processing range of the TIA, and does not address the limits to the total dynamic range of the TIA. Another drawback to this approach is that the TIA will become saturated by the background light as you increase the voltage on the PMT.

[0011] There exists a need for a more efficient cytometry system and method of removing background light from such systems. The present system and method, described herein, allows for the background light in a PMT to be removed from the measurements prior to the conversion of the measured PMT current to a voltage value at the TIA. The system provides a feedback loop that removes the value of the background light measurement before the measured current is converted into a resultant voltage which is subsequently converted to other data. After the value of the background light current is removed, the data measured by the PMT consists only of the pulse measurements and allows the remaining elements of the system to use all of the available computation width on calculating the data.

SUMMARY OF INVENTION

[0012] The invention relates to flow cytometers and systems having an array of processing elements that include the ability to null the background energy entering the detector. The present invention provides a system and apparatus as well as related methods, for efficient measurement of data relating to detected particles, for example, in a flow cytometer or other instruments. It is an object to remove the background light interference from the measured data prior to any manipulation or computation, to allow the processing of the data to concentrate on data relating to the measured pulse only rather than data relating to the combination of the pulse and background light measurements.

[0013] The devices and methods described in more detail below may remove the dynamic range limitation imposed by background light on the Trans Impedance Amplifier (TIA). Even with the most careful optical layout and optical filtration, it is possible that some background light will be present at the input to the PMT. The present system digitally measures the value of the background light DC offset, in realtime, and feeds back the voltage directly to the TIA, releasing the total dynamic range of the TIA. Among other things, removing the background light from the PMT measured data stream prior to conversion at the TIA allows for the elimination of the coupling capacitor and the related baseline restore circuit utilized in the common flow cytometer systems currently employed in the relevant industry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows a schematic diagram of a flow cytometer showing a particle flowing through a sensing zone, where the particle is excited by a beam of light that then causes the particle to fluoresce and/or scatter light.

[0015] FIG. 2 shows a schematic diagram of an example of a detection and conversion system for a flow cytometer according to the present invention.

[0016] FIG. 3 shows the results of certain data characteristics relating to particle size measured by a flow cytometer of the present invention.

[0017] FIG. 4 shows the results of certain data characteristics relating to certain florescent dye or dyes measured by a flow cytometer of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] While this invention is susceptible to embodiments in many different forms, there is shown in the drawings and will herein be described in detailed preferred embodiments of the invention with the understanding that the present disclosure is to be considered as an example of the principles of the invention and is not intended to limit the broad aspect of the invention to the embodiments illustrated.

[0019] As shown in FIG. 1 , a flow cytometer is a device that flows a particle 5 through a sensing zone 10 where the particle is normally excited by a beam of light 15 from a light source 17, such as a laser. The light source 15 causes the particle 5 to fluoresce and/or scatter light. The particles 5 pass through the sensing zone 10 in approximately five microseconds. The fluoresced or scattered light is then separated by filters 20 into portions of the electromagnetic spectrum, usually about twenty nanometers wide, and detected with a photo detector, such as a Photomultiplier Tubes (PMT) 25. The analog pulse is then converted to a digital data stream using Analog to Digital Converters (ADCs) 30. The digital data stream is then processed for its various values by an analysis system 35 or computer. The analysis system 35 is able to generate or extract data relating to the data stream, for example the, signal peak, the signal width, the integral of the signal, or log value of the pulse. This data relates to characteristics of the particles 5. The analysis system 35 can also display the extracted measurement in a form that a user of the system can comprehend.

[0020] Flow cytometers have grown from single parameter instruments to instruments that may have multiple fluorescent parameters along with multiple light scatter and volume parameters. With the addition of each parameter, an additional detector and data conversion channel along with the necessary light separation filter system is added. As shown in the schematic diagram of FIG. 1, five detectors and corresponding data conversion channels are shown.

[0021] Referring now to FIG. 2, a block diagram of a detection and conversion system for a single detector of a flow cytometer is shown. It is understood, however, that a flow cytometer system may contain multiple detectors

and that it may also contain multiple detection and conversion systems associated with those detectors. In FIG. 2, various components are coupled, and signals can pass to the coupled components as described below. In operation, when the flow cytometer's laser pulses light at an object, a PMT 100 detects the fluoresced or scattered pulsed light 60, as well as any constant direct current (DC) or background light 50. The PMT 100 produces a current 1 10 in response to the exposure to the total light, i.e., the background light 50 plus the pulsed light 60. The current 1 10 from the PMT 100 is supplied to the input of a Trans Impedance Amplifier (TIA) 200. The current signal 110 is converted into a single ended voltage signal 230 that is directly related to the value of the current signal 1 10.

[0022] In order to increase the accuracy and reduce the noise of the detection and conversion system, the voltage signal 230 is transmitted to both a low gain branch and a high gain branch. In the high gain branch of the circuitry, the voltage signal 230 outputted from the TIA 200 is transferred to a high gain amplifier 340. The high gain amplifier 340 allows the system to analyze an amplification of the signal 230 within the same sized resolution. The high gain amplifier 340 multiplies the output voltage 230 from the TIA 200 and generates a singled ended amplified voltage signal 350. Amplifying the output voltage 230 may allow for small pulse measurements to be more easily evaluated. Amplifying the voltage signal 230 at this stage also allows voltage signal 230 to be amplified prior to any introduction of noise by other elements of the system. In one example, the high gain amplifier 340 multiplies the TIA output voltage signal 230 by a factor of thirty two. The high gain amplifier 340 and the TIA 200 may both reside on the same chip, to insure gain stability between the TIA output voltage signal 230 and the amplified voltage signal 350 following the voltage amplification by the high gain amplifier 340.

[0023] In the high gain branch, the single ended amplified voltage signal 350 is provided to a differential converter 330. The differential converter 330 may modify the format of the amplified voltage signal 350 to allow for easier conversion by an analog to digital converter (ADC) 420. In a preferred embodiment, the differential converter 330 is a single ended input differential signal converter. The conversion by the differential converter 330 produces a pair of differential amplified signals 370. The resultant format of the signal pair 370 is chosen so that the data stream may be faithfully reconstructed.

[0024] The compatible amplified signal pair 370 is then transferred to differential input low voltage differential signal ADC 420. The ADC 420 is an electronic devices that is able to convert the amplified signal pair 370 to a digital number stream pair 440. Preferably, the ADC 420 implements a continuous digital conversion at rate sufficient to faithfully reconstruct the analog signal. Operating at this rate may produce a sufficient number of data points in a core window for later conversion and analysis.

[0025] Similarly, in the low gain branch, without prior amplification, the single ended voltage signal 230 is provided to a differential converter 320. The differential converter 320 may modify the format of the voltage signal 230 to allow for easier conversion by the analog to digital converter (ADC) 410. In a preferred embodiment, the differential converter 320 is a single ended differential converter. The conversion by the differential converter 320 produces a pair of differential signals 360. The resultant format of the signal pair 360 is chosen so that the data stream may be faithfully reconstructed.

[0026] The compatible single ended signal pair 360 is then transferred to differential input low voltage differential signal ADC 410. The ADC 410 is an electronic device that is able to convert the modified input analog voltage single ended signal pair 360 to a digital number stream pair 430. Preferably, the ADC 410 implements a continuous digital conversion at rate around 20 MHz. Operating at this rate may produce a sufficient number of data points in a core window for later conversion and analysis.

[0027] The use of serial output ADCs 410, 420 rather than parallel output analog to digital converters may also allow both ADCs 410, 420 to reside on the same chip. Having the ADCs 410, 420 on the same chip, may help maintain gain stability between the digitally converted data stream pairs 430, 440 following the conversion from the analog realm to the digital realm by the ADCs 410, 420. The use of high speed serial ADCs 410, 420 also may allow for high circuit densities in design which decreases the size of the system and saves total cost of manufacturing.

[0028] After the signal pair 360 and the amplified signal pair 370 are converted to a digital signal pair 430 and an amplified digital signal pair, 440 by the ADCs 410, 420, the separate signal pairs 430, 440 may be provided to and received

by a logic element, shown as a field programmable gate array (FPGA) 500. The FPGA 500 deserializes the serial data stream then calculates certain signal traits, such as the signal peak, the signal baseline, the signal width and the integral of each the separate signal pairs 430, 440. The use of the FPGA 500 to integrate the separate signal pairs 430, 440, also makes it possible to achieve greater than twenty four bits of integral data from a single fourteen bit analog to digital converter without stitching two analog to digital converters together.

[0029] Following this determination of the signal traits, the FPGA 500 transmits the signal traits 520 separately for each signal pair 430, 440 via a parallel digital 8 bit wide Universal Serial Bus (USB) connection to an analysis system 700 for analysis. In a preferred embodiment, the analysis system 700 is a computer system which may be a special purpose computer, or a general purpose computer with analysis and data generation software. The analysis system 700 then analyzes the signal traits 520 and has the ability to generate data such as background light data 530, which can be used to modify the performance of the detection and conversion system . The data 530 can be provided to the FPGA 500 over the same USB connection as described above.

[0030] As described above, when measuring a particle, the laser light is pulsed and the sensing zone of the PMT 100 receives and thus measures both the DC background light 50 and the pulsed light 60. It is therefore preferred that the unwanted background light component 50 be removed prior to the modification of the voltage signal 230, leaving only the pulse signal 60 component of the voltage signal 230. Accordingly, in the present system, prior to the laser or pulsed light signal 60 becoming active, the system operates with only the DC background light component 50 present in the system. Thus only the background light 50 may be initially measured by the PMT 100. As described above, the signal 110 is an input to the TIA 200 and the signal is manipulated the same as described above. However, after the signal traits 520 are measured by the FPGA 500, and the signal traits 520 are transferred to the analysis system 700 using the USB connection, the analysis system 700 generates background light data 530 and transmits the data to the FPGA 500.

[0031] Taking advantage of the pulse signal's inactivity, the analysis system

700 is able to determine the value of the background light offset 50 alone. The

analysis system 700, having received the signal traits 520 of only the background light component, may calculate data relating to an input modification to the TIA 200 required to offset the background light interference 50. For example, the system 700 may calculate the root mean square (RMS) value of the background light and provide data 530 to the FPGA 500 relating to this value. The FPGA 500, based on this data or instructions from the analysis system 700, forwards digital offset signal data 510 to the digital to analog converter (DAC) 600. The digital offset signal data 510 is preferably a composite of multiple readings of the background light value, and for example may be the average of 1024 different measurement cycles. The DAC 600 then converts the digital signal 510 to an analog null signal 610. The null signal 610 is then relayed from the DAC 600 to the TIA 200 via a feedback loop. The TIA 200 is able to receive the analog null signal 610, and remove the value of the null signal 610 from future conversions of the input current 110 to the voltage signal 230.

[0032] After this initialization process is complete, and the background light value 50 has been measured by the analysis system 700, and the null signal 610 has been provided to the TIA 200, the pulsed signal 60 may also be activated. The result is that the null signal 610 is removed from the input current 110 by the TIA 200 and the TIA 200 only converts the pulsed signal 60 component of the input current 110. The resultant voltage signal 230 may be free from any component relating to background light value 50 or interference. Because the value of the background light offset component of the input current 110 is removed by the TIA 200, any future modifications by the amplifier 340, the converters 320, 330, the ADCs 410, 420 occurring to the voltage signal input 230, only affect the pulsed signal component 60 of the input current 110. Thus the dynamic range of the TIA 200 may be extended to its maximum theoretical value. The removal of the background light component 50 at such an early stage in the process, may also eliminate the need for any baseline restoration circuit.

[0033] The resultant data resolution allows for the analysis of the entire inherent dynamic range of the detector 100 by the analysis system 700. The background DC offset's removal no longer has a lasting negative effect on the analysis of the data relating to the pulsed light component 60. The analysis system may generate data about different characteristics of the particles depending on the detector used (e.g., forward scatter, side scatter, or 525 nm, 578 nm or 620 nm

fluorescent detectors). As shown in FIG. 3 data relating to particle size from a forward scatter detector can be generated and displayed on a monitor, or other output device. In FIG. 3, the vertical axis relates to particle size and the horizontal axis relates to the number of particles.

[0034] As shown in FIG. 4, data relating to particles and a certain fluorescent dye or dyes is shown. In FIG. 4, the vertical axis relates to the number of particles and the horizontal axis relates to the number of molecules that contain dyes in the orange region. The detector used for the example may be a 578 nm detector that is 50 nm wide. As shown, the horizontal axis displays six decades of linear dynamic response data.

[0035] Of course other size and types of detectors may be used, as is known, to measure other characteristics such as side scatter and fluorescence in other regions, e.g. green, red or dark red. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.