WO2000005566A1 - Flow cytometer droplet break-off location adjustment mechanism - Google Patents
Flow cytometer droplet break-off location adjustment mechanism Download PDFInfo
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- WO2000005566A1 WO2000005566A1 PCT/US1999/014284 US9914284W WO0005566A1 WO 2000005566 A1 WO2000005566 A1 WO 2000005566A1 US 9914284 W US9914284 W US 9914284W WO 0005566 A1 WO0005566 A1 WO 0005566A1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2015/1406—Control of droplet point
Definitions
- the present invention relates in general to flow cytometer systems, and is particularly directed to a new and improved flow cytometer architecture and signal processing control mechanism therefor, that is operative to monitor prescribed characteristics of non- sorted and deflected droplet streams, and to controllably adjust drop-formation and drop-sorting deflection parameters, so as to maintain the point at which droplets break off from the cytometer ' s fluid stream at a calibrated droplet break-off location.
- Flow cytometers are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease.
- a patient's body fluid e.g., blood cells
- such instruments may be used to sort and collect healthy blood cells (stem cells) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell reproduction.
- particles 11 to be analyzed such as cells of a centrifuged blood sample stored in a container 11, are injected into a (pressurized) continuous or uninterrupted stream of carrier fluid (e.g., saline) 12.
- carrier fluid e.g., saline
- This carrier fluid stream is directed along a flow channel 13 of a fluid flow chamber or cell 14.
- the fluid flow channel 13 is intersected at a location 15 by an output beam 16 emitted by an optical illumination subsystem, such as one or more lasers 17.
- an optical illumination subsystem such as one or more lasers 17.
- Located optically in the path of the laser output beam 16 after its being intercepted by the carrier fluid stream are one or more photodetectors of a photodetector subsystem 20.
- the photodetector subsystem 20 is positioned to receive light modulated by the contents of (particles/cells within) the fluid stream, including light reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell.
- the fluid flow channel 13 through the cytometer flow chamber is configured and sized to pass the particles or cells only one cell at the time through the intersection location 15 with the laser's output beam 16.
- each modulation signal such as that shown at 21 and occurring at a time tO in the timing diagram of Figure 2 can be associated with an individual cell. If the output of the photodetector subsystem 20 satisfies prescribed ' sort ' criteria associated with one or more parameters of a desired cell, it is used to control the sorting of a droplet 23 of carrier fluid containing that cell by an electrostatic droplet sorter 24 located downstream of an exit port or aperture 18 of the flow chamber .
- the carrier fluid stream is converted into individual droplets by an acoustically (e.g., piezoelectric transducer) driven droplet generator 27, which is coupled to the fluid flow chamber.
- the individual droplets do not form immediately at the exit port 18 of the fluid flow chamber, but proceed as an interconnected droplet stream 22 and break off at a location 25 downstream of the chamber exit port.
- the location 25 at which the droplets form downstream of the flow chamber exit port 18 is adjusted by varying the parameters of the droplet generator drive signal.
- the rate at which droplets are formed is governed by the frequency of the acoustic drive signal, and the droplets become synchronized with the frequency of the piezo vibration of the droplet generator 27.
- the acoustic drive frequency applied to the droplet generator 27 may be on the order of from four to one hundred Khz, at a fluid pressure on the order of from three to seventy psi.
- the photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor of the cytometer 's control workstation 50. Based upon this analysis, the control processor supplies control signals to a charging and deflection control circuit 52 of the droplet sorter 24 to sort or abort the droplet.
- the droplet sorter 24 employs an electrostatic charging collar 31 surrounding the travel path 26 of the droplet sequence.
- Charging collar 31 may comprise a metallic cylinder that is located so as to surround the location along the droplet sequence travel path 26 where the individual droplets 23 separate from the fluid stream, and is typically several droplets in length.
- the charging collar 31 is positioned vertically downstream of the fluid chamber exit port 18 and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates 33 and 35 between which the stream of charged droplets 23 pass as they travel downwardly and are either sorted along a sort path 36 into a sorted droplet collection container 41, or allowed to pass unsorted along travel path 26 into an aborted or discarded waste container 43.
- a prescribed charging voltage pulse 32 of a duration tl2 is selectively applied to the charging collar 31 at time tl, i.e. at the end of the sort delay tOl, and terminating at time t2 at the end of the pulse duration interval tl2, thereby charging a droplet 23C that should contain the cell to be sorted.
- the selectively charged droplet 23C passes between the two opposite polarity high voltage deflection plates 33 and 35, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge.
- This electrostatic steering action directs the charged droplet 23C along a deflected travel path 36 on one side of the main droplet travel path 26, and into the sorted droplet collection container 41.
- Sort delay is affected by various parameters including the pressure of the carrier fluid, size and surface characteristics of the droplet generator exit port, the viscosity of the carrier fluid, and the amplitude of the piezo vibration. While some parameters, such as the pressure of the fluid carrier, which affect the position of the droplet formation point, can be controlled with precision, others cannot be controlled.
- material may build up on the flow chamber exit port, causing a change in the natural energy of the fluid stream, and moving the droplet formation point closer to the flow chamber.
- Other factors include acoustic coupling of the instrument vibration, room noise, vibration in the room machinery external to the unit, and so on.
- this may be accomplished by initially manually setting the droplet formation point 25 at some predetermined distance from the laser intersection point 15, using a precision imaging aid (such as a microscope objective or a video camera) to observe the fluid steam.
- a precision imaging aid such as a microscope objective or a video camera
- Strobing a light emitting diode in sync with the excitation frequency of the piezoelectric drive signal to the droplet generator 27 will make the droplets 23 formed from the fluid stream appear to be stationary. Then, by controllably increasing or decreasing the amplitude of the piezoelectric drive signal, the operator can move the droplet formation point closer or farther away from the laser intersection point, until the point at which the drops first form coincides with a reference or positioning mark. Next, the operator inputs to the sorting system a sort delay time that has been determined on the basis of previous experimentation, so as to place the system within several drops of the actual sort delay time.
- the operator sets up and runs a calibration sort operation, using test beads, which mimic biological cells in terms of size.
- the beads are sorted onto a slide, and the slide is observed (under a microscope) to determine whether the number of beads on the slide coincides with the number of beads the system reported as having sorted.
- the system is adjusted by changing the sort delay time, or by moving the droplet formation point by varying the amplitude of the acoustic drive signal. This operation is iteratively repeated as necessary until the beads counts are correct. With the system thus initially calibrated, it may then be monitored visually for drift, with the operator observing the fluid stream and droplets for movement. To verify that the sort parameters remain the same, the slide and bead analysis sequence described above may be repeated. It will be readily appreciated that this trial and error procedure is a time consuming process, and sample may be lost or the sort container contaminated during the sorting process without operator knowledge .
- test mode optical forward error correction system comprised of an additional laser - photodetection subsystem, that takes a ' second look' into the continuous fluid stream at some point downstream of the laser beam intersection location 15, but prior to the droplet break off point 25.
- the purpose of the second optical system is to confirm that test beads that have been injected in the fluid stream arrive at the downstream detection location at a time that they are expected.
- an auxiliary laser is employed to determine whether there has been a shift in the overall velocity of the droplet stream.
- An obvious shortcoming of this approach is that it does not address the fundamental problem of determining exactly where the last attached droplet breaks off from the fluid stream.
- a further proposal places a second laser at an initially established droplet break off point and then monitors the stream at that point. Unfortunately, since the laser is fixedly positioned, it cannot be readily repositioned if the break off point moves.
- the above-discussed drawbacks of conventional flow cytometer instruments are successfully remedied by a feedback-based signal processing mechanism that is operative to maintain the droplet break-off point at an initially calibrated spatial location (within the droplet charge collar of a droplet sorting mechanism) .
- the invention looks for gaps in the fluid droplet stream that have been created by the deflection of charged droplets. The difference between the times at which these gaps are detected at a prescribed downstream location in the path of the droplet stream and the times at which deflected droplets that created the gaps were charged at the droplet charge collar is compared with a calibration reference interval.
- the instrumentation architecture of a flow cytometer system employing the droplet break-off location adjustment mechanism in accordance with the present invention augments the system of Figure 1, described above, with an unsorted droplet 'gap' detector associated with the main or unsorted droplet travel path, and a deflected/sorted droplet detector associated with the sorted droplet travel path.
- the unsorted droplet gap detector comprises an optical energy source and an associated optical detector which are positioned to provide a viewing window that intersects the unsorted droplet travel path at a location downstream of the droplet sorter.
- the unsorted droplet gap detector is operative to identify the presence of a gap in what is otherwise a generally spatially periodic sequence of unsorted (uncharged) droplets that have broken off from the carrier fluid stream at a location within the droplet sorter's charging collar and are traveling downwardly toward a waster container.
- the presence of a gap in the unsorted droplet stream indicates that a (to be sorted) droplet has been charged and is traveling along a deflection path toward a sorted droplet collection container.
- the difference between the time at which a sorted/charged droplet breaks off from the fluid stream exiting the cytometer flow chamber and a subsequent time at which the gap in the droplet stream resulting from the deflection of the charged droplet arrives at the gap detector is a prescribed 'gap' transit time interval.
- this gap transit time will remain constant. However, any change in the gap transit time will indicate that the droplet formation point has moved from its calibration point.
- the change in the gap transit time is employed to adjust the amplitude of the piezo drive signal to the droplet generator, so as to bring the instrument back into calibration.
- a 'predicted' gap transit timer is started at the termination of the sort delay, which is also coincident with the time at which a charging pulse is applied to the charging collar of the droplet sorter.
- the predicted gap transit timer is programmed to time out at a time which occurs prior to the time at which the gap is expected to arrive within the viewing window of the unsorted droplet gap detector. This time is set equal to the calibrated gap transit time interval, minus the length of time required for a droplet that is a prescribed number of (e.g., two) droplet locations upstream from the gap detector to reach the gap detector.
- each of a gap detector prediction timer and a gap prediction difference timer is started.
- the gap detector prediction timer times out over a duration equal to some number N of droplet periods, which corresponds to the time required for N consecutive unsorted drops to travel past a given point along the unsorted droplet travel path.
- the time at which the gap detector prediction timer times out occurs slightly later than the time required for a droplet to travel from a position upstream of the gap detector's viewing window to a position downstream of that position, so that the gap detector prediction timer's timing window or interval is sufficient to cover a droplet travel distance that covers the entire width of the gap detector viewing window.
- the gap prediction difference timer has a timing duration that begins at the end of the predicted gap transit timing window and terminates at the time at which the gap is detected by the unsorted droplet gap detector. Namely, the sum of the durations of the predicted gap transit window and the gap prediction difference window is equal to the calibrated gap transit time.
- the output of the gap-detector station's optical detector is monitored for a signal transition - indicating the presence of a gap in the unsorted droplet steam.
- the occurrence of this gap detection signal at a time other than the expected (calibrated) time indicates a timing error, the value of which is equal to the measured value of the gap prediction difference timer minus the offset droplet periods .
- a change in the gap transit time interval means that the droplet formation location has moved farther away (downstream) from or closer (upstream) to the exit port of the fluid flow chamber, and has caused a variation in the sort delay time interval.
- the amplitude of the piezo drive signal to the droplet generator is changed accordingly, so that droplets will break off at the calibrated point and thereby reduce the currently detected gap transit time interval into alignment with the calibrated interval. Because the droplets travel through air between the exit port of the fluid flow chamber and the droplet collection containers, they encounter air resistance which affects the pattern of the droplets, and thereby interferes with gap timing.
- air resistance In addition to affecting the travel of gaps along the unsorted droplet travel path, air resistance also retards the travel of deflected droplets along the sorted droplet deflection path. Although this air resistance is not a problem along the sorted travel path if the droplets being deflected/sorted are spaced apart from one another by undeflected droplets, it becomes a problem if the droplets being deflected from the unsorted travel path are immediately consecutive to one another. Where two immediately consecutive droplets are deflected from the main travel path, the speed of the forwardmost droplet is retarded by the encountered air resistance, causing it to form a droplet pair or packet with the next consecutive and faster moving deflected droplet.
- the detector sees what appears to be a single large droplet and therefore generates a single output pulse having an amplitude that is larger than in the case of a single droplet.
- the sorted droplet detector is employed to control the magnitude of the charging voltage pulse applied to the charging collar of the droplet sorter, so that the travel path of the sorted droplet will remain coincident with a droplet receiving opening into the sorted droplet collection container.
- the resistance of the air retards the speed of the first two droplets so that they form a droplet trio packet with the next consecutive and faster moving third deflected droplet.
- this droplet trio packet passes the sorted droplet detector, the droplet detector again sees what appears to be a single large droplet and therefore generates a single pulse having an amplitude that is larger than in the case of a single droplet or a droplet pair.
- the fourth and any additionally consecutive droplets will not be effectively retarded by air resistance. It has been observed that where a droplet is preceded by three or more droplets, it travels unretarded and is spaced apart from an upstream droplet or packet of droplets. As a consequence, for more than three consecutive sorted droplets, the sorted droplet detector will detect an initial trio packet as a single large droplet followed by the fourth and any subsequent droplets as normal sized, individual droplets .
- each output pulse is seen to represent only one deflected droplet.
- sorting signals are counted by a sorting signal counter, the output of which is compared with a running count of the number of pulses produced by the sorted droplet detector. If the difference between the two count totals exceeds a prescribed error limit, the magnitude of the charging voltage pulse applied to the charging collar of the droplet sorter is adjusted until _ the two compared droplet count values are the same. At this point the magnitude of the charging voltage applied to the droplet sorter's charging collar will be the value that causes the deflection travel path of the sorted droplets to be coincident with the opening into the sorted droplet collection container, thereby maximizing collection of all sorted droplets. Should adjustment of the charging voltage fail to bring the droplet count value difference within tolerance, an alarm condition is declared, terminating the sorting process until the system is recalibrated.
- a transparent protective chamber is used to isolate the droplet travel region between the fluid flow chamber and the collection containers from the movement of ambient air, such as may be caused by motion of a system operator in the vicinity of the cytometer.
- an isolation chamber is effective to shield the droplet travel paths of the cytometer from the entry of potentially disturbing air currents from the surrounding ambient
- a problem associated with the use of such an enclosed housing is the fact that small fluid particles created when the droplets are formed may deposit on the interior surfaces of the chamber and obstruct the sensing regions of the gap detector and the sorted droplet detector.
- the substantial saline humidity may reduce the electrostatic breakdown potential between the deflection plates of the droplet sorter.
- a pair of vacuum- controlled air curtains are directed along the interior wall surfaces of the isolation chamber. Because the air curtains flow only along the wall surfaces of the chamber, they do not interact with or affect the velocity or the direction of travel of the non-sorted or sorted droplets.
- the charging voltage pulse In order to properly charge a droplet for deflection, the charging voltage pulse must be applied to the charging collar during the time that the droplet is still connected to or part of the fluid stream (as the last connected droplet) , in order to ensure that a conductive path is available for charge transfer. Also, the charging voltage must be maintained until the droplet breaks off from the main fluid stream. The droplet will carry this charge until it comes in contact with a conductive surface, allowing the charge to dissipate off the droplet.
- the charging voltage pulse typically has a pulse width equal to one droplet period.
- the charging voltage pulse is terminated prior to the end of a normal droplet period, so as to ensure that a droplet being charged is still in the process of breaking off from the main carrier fluid stream at the calibrated sorting time. If the droplet break-off time drifts outside of this drop-charging window, then the droplet will be uncharged when it breaks off from the main carrier stream, so that it will not be deflected and leave a gap in the unsorted droplet stream. Although not sorting the droplet will be detected as a sorting error, the break-off location drift that caused the error will not allow an undesirable droplet to be charged and sorted) , thereby avoiding contamination of the contents of the sorted droplet collection container.
- Figure 1 diagrammatically illustrates the general instrumentation architecture of a flow cytometer
- Figure 2 is a timing diagram associated with the operation of Figure 1;
- Figure 3 diagrammatically illustrates the general instrumentation architecture of a flow cytometer system employing the droplet formation location adjustment mechanism in accordance with the present invention;
- Figure 4 is a timing diagram associated with the operation of Figure 2 ;
- Figure 5A and 5B are respective timing diagrams illustrating the manner in which the travel time of a gap in a droplet stream may be measured;
- Figure 6 diagrammatically illustrates the manner in which air resistance retards droplet travel speed;
- Figure 7 diagrammatically illustrates two immediately consecutive deflected droplets forming a droplet pair packet
- Figure 8 diagrammatically illustrates three immediately consecutive deflected droplets forming a droplet trio packet
- Figure 9 shows four immediately consecutive deflected droplets forming a droplet trio packet followed by an individual droplet
- Figure 10 diagrammatically illustrates the comparison of sorting signals counted by a sorting signal counter with droplet count signals
- Figures 11, 12 and 13 diagrammatically illustrate an optically transparent air flow-constraining protective chamber
- Figure 14 diagrammatically illustrates the manner in which a reduced magnitude droplet charging pulse causes a selected empty droplet to be deflected along an auxiliary travel path
- Figure 15 shows a side view of a charging collar
- Figure 16 is a perspective view of the optically transparent air flow-constraining protective chamber of Figures 11 - 13;
- Figures 17 and 18 are top views of a charging collar;
- Figure 19 is a front view of a charging collar;
- Figure 20 diagrammatically illustrates air flow curtains within the optically transparent air flow- constraining protective chamber of Figure 11.
- FIG. 3 diagrammatically illustrates the instrumentation architecture of a flow cytometer system employing the droplet break-off location adjustment mechanism in accordance with the present invention, but rotated by 90°, in order to facilitate its association with respective timing diagrams, to be described.
- inventive cytometer architecture comprises essentially the same components as shown in Figure 1, described above, but with an additional 'post-charging' droplet gap-monitoring station 60.
- This additional droplet gap-monitoring station is diagrammatically illustrated as comprising an optical energy source 61, such as an infrared emitter, and an associated optical detector 63, which are positioned to provide a viewing window 65 that intersects the droplet travel path 26 at a location downstream of the electrostatic deflection plates 33 and 35 of the droplet sorter 24.
- an optical energy source 61 such as an infrared emitter
- an optical detector 63 which are positioned to provide a viewing window 65 that intersects the droplet travel path 26 at a location downstream of the electrostatic deflection plates 33 and 35 of the droplet sorter 24.
- the function of the droplet gap-monitoring station 60 is to identify the presence of a gap 28 in what is otherwise a generally spatially periodic sequence of unsorted (uncharged) droplets 23 that have broken off from the carrier fluid stream 22 at a location 25 within the charging collar 31 and are traveling downwardly toward waste container 43.
- the presence of a gap 28 in the droplet stream indicates that a droplet has been charged and is traveling along deflection path 36 toward the sorted droplet collection container 41.
- the fluid pressure of the carrier stream is held constant by a separate fluid pressure control system (not shown) , so that the velocity of the droplet stream does not change .
- this gap transit time interval tl3 will remain constant. However, any change in the length of the transit time interval tl3 will indicate that the droplet formation point has changed from its calibration point.
- an increase in the gap transit time interval tl3 means that the droplet formation location 25 has moved closer (upstream) to the exit port of the fluid flow chamber, and has thereby caused the drop (containing the particle which is qualified to be sorted) to break off from the fluid stream, before the sort pulse 31 is applied. This will cause the desired particle to travel to the waste container 43, instead of the collection container 41. If the time drifts by a whole drop, then the drop following will be sorted instead, which could contaminate the contents of the collection container 41 with whatever that droplet contained.
- the amplitude of the piezo drive signal to the droplet generator 27 is reduced, causing less vibrational energy to be coupled to the fluid stream, so that the droplets break off farther downstream, and bring the gap transit time interval tl3 back into calibration.
- a decrease in the gap transit time interval means that the droplet formation location 25 has moved farther away
- the amplitude of the piezo drive signal to the droplet generator 27 is increased, causing more vibrational energy to be coupled to the fluid stream, so that droplets to break off farther upstream (closer to the fluid chamber's exit aperture), bringing the gap transit time interval back into calibration.
- sort representative signals are written to sequential locations of a memory device at a rate equal to the resolution required for the sorting operation.
- a write clock of 256 Khz associated with a resolution of one-sixteenth of a droplet (when the piezo-electric transducer is driven at 16 KHz) , may be employed.
- the stored sorting data which is to be compared with signals generated from downstream detection circuitry, is then read out of an offset location in the memory device, which corresponds to a delay of one transit period from the droplet sorter 24 to the detection circuitry.
- the transit time is divided by 1/256,000 to determine the offset.
- the delayed sorting information signals are effectively made concurrent with the real time detection signals from the gap and drop detectors.
- the data is then analyzed to determine if the events of interest are qualified, and the difference between the signals is written to memory for processing by the system processor.
- a predicted gap transit interval tl4 is measured from the break-off/charging time tl to time t4, which occurs prior to the time t3 at which the gap 28 is expected to arrive at the droplet gap-monitoring station 60.
- the time t4 at which the predicted gap transit timing window tl4 terminates is set to be equal to the calibrated gap transit time interval tl3 , minus the length of time required for a droplet that is a predetermined number (e.g., two as a non-limiting example) of droplet locations upstream from the gap- monitoring station to reach the gap-monitoring station.
- each of a gap detector prediction timing measurement interval t45 and a gap prediction difference timing measurement interval t43 is started.
- the gap detector prediction timing measurement interval t45 times out over a duration equal to some number N of droplet periods (e.g., four droplet periods) , which corresponds to the time required for N (e.g., four) consecutive drops to travel past a given point along the droplet travel path 26.
- the time t4 at which the predicted transit interval tl4 terminates, is two droplets upstream of the front end 67 of the viewing window 65 of the gap- monitoring station 60
- the time t5 at which the gap detector prediction timing measurement t45 times out is two droplet periods downstream of the upstream end 67 of the viewing window 65 of the gap-monitoring station 60.
- the time t5 occurs slightly later than the time required for a droplet to travel from a position two droplets upstream of the front end 67 of the viewing window 65 to a position four droplets downstream of that position, so that the timing window of gap detector prediction timing measurement t45 is sufficient to capture the actual gap as it passes the front end 67 of the viewing window 65, and to do so even if the gap has drifted by more than one drop.
- the gap prediction difference timing measurement window t43 has a timing duration that begins at time t4 at the end of the predicted gap transit timing window tl4 and terminates at the time at which the leading edge of gap 28 is detected at the droplet gap- monitoring station 60. Namely, the sum of the durations of the predicted gap transit timing window tl4 and the gap prediction difference timing window t43 is equal to the actual gap transit time interval.
- the output of the gap-detector station's optical detector 63 is monitored for a signal transition 200 - indicating the presence of a gap in the droplet steam in travel path 26.
- Figure 5A shows a gap transition with no error, where the gap occurs at time t3 , which is the end of the calibrated transit time tl3.
- one-half of the gap detector prediction timing window t45 is subtracted from the gap prediction difference t43, which in this case leaves zero, or no error.
- a gap detection signal may occur at time t3 ' , which is earlier (e.g., by one droplet period), than the time t3 at which the gap signal should occur if the system were in calibration.
- the actual timing error shown in Figure 5B as a gap prediction error
- the actual timing error is equal to the measured value of the gap prediction difference timing window t43 minus one-half of the gap detector prediction timer window, or two droplet periods .
- a decrease in the gap transit time interval tl3 means that the droplet formation location 25 has moved farther away (downstream) from the exit port of the fluid flow chamber, and has caused the plug of particle carrying fluid to become misaligned with the sort delay time, causing an uncertainty as to the location of the particle within a particular drop, thereby degrading the recovery of those particles in the sort collection container, as well as the purity.
- the amplitude of the piezo drive signal to the droplet generator 27 will be slightly increased, so that droplets will break off farther upstream (closer to the fluid chamber's exit aperture), and thereby reduce the currently detected gap transit time interval tl3 ' towards alignment with the calibrated interval tl3.
- the droplets travel through air between the exit port 18 of the fluid flow chamber 14 and the droplet collection containers 41 and 43, they encounter air resistance which affects the pattern of the droplets, and thereby interferes with gap timing.
- those droplets which have no droplets directly in front of them will encounter sufficient air resistance as to decrease their speed and cause them to fall back or be retarded slightly from their expected positions.
- droplets which have some number of droplets (e.g., three or more) directly in front of them will not encounter such air resistance, but will maintain their speed along their travel path.
- the gap measurements derived by the various timing measurements for droplets traversing the main or unsorted droplet travel path 26 are not employed unless the gaps are immediately preceded by a prescribed number of non-sorted droplets (e.g., three or more) .
- air resistance also retards travel of deflected droplets along sorted droplet deflection path 36. This is not a problem if droplets deflected from unsorted droplet travel path 6 are spaced apart from one another by undeflected droplets (namely immediately consecutive droplets in the unsorted droplet travel path are not deflected) . However, it is a problem if droplets deflected from unsorted droplet travel path 26 are immediately consecutive to one another.
- Figure 7 diagrammatically illustrates the condition where two immediately consecutive droplets 23D1 and 23D2 are deflected from the unsorted droplet travel path. Because of the resistance of the air, the speed of the forwardmost droplet 23D1 is retarded, causing it to form a droplet pair packet 23P2 with the next consecutive and faster moving deflected droplet 23D2.
- a droplet pair packet 23P2 intersects an optical beam generated by a light source 68, such as an IR emitter, the output of which is directed upon a deflected or sorted droplet detector 70, detector 70 sees what appears to be a single large droplet and therefore generates a single pulse 71 having an amplitude that is larger than the case of a single droplet.
- sorted droplet detector 70 is employed to control the magnitude of the charging voltage pulse 32 applied to the charging collar 31, so that the travel path 36 of the sorted droplet will remain coincident with the opening into the sorted droplet collection container 41, thereby maximizing collection of all sorted droplets.
- Figure 8 diagrammatically illustrates the condition where three immediately consecutive droplets 23D1, 23D2 and 23D3 are deflected from the unsorted droplet travel path. Again, the resistance of the air retards the speed of the first two droplets 23D1 and
- FIG. 9 shows four immediately consecutive droplets 23D1 - 23D4 being deflected from the unsorted droplet travel path.
- the first three droplets 23D1- 23D3 form a trio packet 23P3 that travel at the speed of the third droplet 23D3.
- the fourth droplet 23D4 is preceded by three or more droplets, it travels unretarded and spaced apart from the trio packet 23P3.
- the sorted droplet detector 70 sees the trio packet 23P3 as a single large droplet followed by the fourth droplet 23D4, and therefore generates a first pulse 73 having a relatively large amplitude, followed by a second pulse 74 having an amplitude representative of a single droplet . Because the output pulses from the sorted droplet detector 70 are not discriminated as to size, each output pulse is seen to represent only one deflected droplet. To correct for the effect of the air resistance 'packetizing' of pairs and trios of droplets on the pulses generated by the sorted droplet detector 70, a determination is made as to whether the sort (droplet-charging) signals that are incrementally applied to the droplet sorter 24 are associated with sequential droplets.
- sorting signals As shown in Figure 10, as these sorting signals are generated they are counted by a sorting signal counter 75, the output of which is compared in a comparator 77 with the output of a counter 78, which counts the pulses produced by the sorted droplet detector 70. If the difference between the two count totals exceeds a prescribed error limit, the magnitude of the charging voltage pulse 32 applied to the charging collar 31 of the droplet sorter 24 is adjusted by the workstation processor 50 until the two compared droplet count values are the same .
- the magnitude of the charging voltage applied to the droplet sorter's charging collar will be the value that causes the deflection travel path 36 of the sorted droplets to be coincident with the opening into the sorted droplet collection container 41, thereby maximizing collection of all sorted droplets. If adjustment of the charging voltage fails to bring the droplet count value difference within tolerance, an alarm condition is declared, terminating the sorting process until the system is recalibrated. In addition to the problem of retarded speed caused by the resistance of the air through which both sorted and non-sorted droplets fall, there is an ancillary problem of effects of unwanted air currents in the sorting area. In particular, the gap timing adjustment mechanism described above is sensitive to even very small fluctuations in movement of the ambient air around the droplets .
- the protective chamber is optically transparent and may be configured in the manner shown in Figures 11-13 and 15-20, as a generally conically rectilinear housing 100 of a sturdy transparent material, such as a clear plastic material, having a pair of sidewalls 102 and 104, which diverge from an inlet port 106 and terminate at an endwall 108, so as to define an open interior region 110 therebetween. Top and bottom surfaces of the housing perimeter are covered by respective transparent cover plates 112 and 114.
- the charging collar 202 is configured as a cube-shaped plastic piece with a passage 205 through it, to allow the droplets exiting the flow cell 204 at orifice 206 to pass through to the chamber inlet 106.
- the charged droplet deflection plates 33 and 35 are mounted alongside the exterior of sidewalls 102 and 104, as shown.
- the center of the endwall 108 has a generally longitudinal bore 121 that is aligned with the unsorted droplet travel path 26 and is coupled via an exhaust port 123 to waste collection container 43.
- a pair of sorted droplet collection ports 131 and 133 are disposed at portions of the endwall 108 offset to either side of the bore 121.
- the ports 131 and 133 are valved with stop-cocks to prevent contamination of the chamber by external unfiltered air, as well as to prevent biological material that may be left within the chamber after sorting hazardous particles from contaminating the instrument. These stop-cocks are closed by the operator when the collection tubes are removed.
- the sorted droplet collection container 207 is coupled to one of these droplet collection ports.
- the use of an enclosed housing is subject to the fact that small fluid particles created when the droplets are formed may deposit on the interior surfaces of the chamber and obstruct the sensing regions of the gap detector and the sorted droplet detector.
- the substantial saline humidity may reduce the electrostatic breakdown or shorting potential between the deflection plates 33 and 35.
- 211, 213 are directed from the charging collar and from the ports 141, 143 along the interior wall surfaces of the isolation chamber.
- the chamber air inlet ports 141 and 143, as well as the charge collar inlet ports 203 and 208, are connected to filters designed to prevent the passage of particles two microns or larger.
- the filters are ported to the ambient air.
- the air curtains flow only along the wall surfaces of the chamber, they do not interact with or affect the velocity or the direction of travel of the non-sorted or sorted droplets.
- the controlled air curtains are exhausted by way of low vacuum port 201.
- the vacuum level is set such that the air curtains are pulled through the chamber, but not so high that the air curtains interfere with the gap timing measurements.
- a vacuum of one-half to one inch of mercury may be employed.
- relatively long time intervals may occur between sorted droplets, so that output signals from the gap detector 60 and the sorted droplet detector 70 are ostensibly unavailable for conducting the on-line system adjustments described above.
- the middle one of a trio of consecutive droplets is 'slightly' charged by applying a reduced magnitude voltage (e.g., one having only ten percent of the normal magnitude of the charging voltage pulse) to the charging collar 31.
- a reduced magnitude voltage e.g., one having only ten percent of the normal magnitude of the charging voltage pulse
- this reduced magnitude charge causes the selected empty droplet 23E to be deflected along an auxiliary travel path 46, that is slightly off to the side of the unsorted droplet travel path 26, but still allowing the droplet 23E to be collected by the unsorted droplet collection container 43.
- This does not allow for checking the position of normally deflected droplets; however, if the deflection field voltage applied to plates 33 and 35 has degraded, the ten percent voltage will not be sufficient to cause a detectable gap 28 in the unsorted droplet stream, so that the deflection angle is verified as not having degraded.
- the charging voltage pulse 32 In order to properly charge a droplet for deflection, the charging voltage pulse 32 must be applied to the charging collar 31, while the droplet is still connected to the fluid stream 22 (as the last connected droplet) , in order to ensure that a conductive path is provided for charge transfer. In addition, the charging voltage must be maintained until the droplet breaks off from the fluid stream. The droplet will carry this charge until it comes in contact with a conductive surface, allowing the charge to dissipate off the droplet.
- the charging voltage pulse typically has a pulse width equal to one droplet period.
- the width of the charging voltage pulse 32 is reduced to some fraction of a normal droplet period (e.g., thirty percent, as a non-limiting example) . If the droplet break-off time drifts outside of this drop- charging window, then the droplet will not have any charge when it breaks off from the carrier stream, so that it will not be deflected and leave a gap 28 in the unsorted droplet stream.
- the invention is operative to monitor prescribed characteristics of deflected droplet streams, and to controllably adjust drop-sorting deflection parameters, so as to maintain the deflected travel path of sorted droplets coincident with the opening into a sorted droplet collection container, thereby maximizing collection of all sorted droplets.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99931885A EP1099105B1 (en) | 1998-07-20 | 1999-06-25 | Flow cytometer droplet break-off location adjustment mechanism |
JP2000561483A JP4426106B2 (en) | 1998-07-20 | 1999-06-25 | Apparatus and method for controlling droplet separation point of flow cytometer |
DE69929792T DE69929792T2 (en) | 1998-07-20 | 1999-06-25 | MECHANISM FOR ADJUSTING THE DRIP RIPPING POINT IN A FLOW CYTOMETER |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/119,368 | 1998-07-20 | ||
US09/119,368 US6079836A (en) | 1998-07-20 | 1998-07-20 | Flow cytometer droplet break-off location adjustment mechanism |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000005566A1 true WO2000005566A1 (en) | 2000-02-03 |
Family
ID=22384040
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/014284 WO2000005566A1 (en) | 1998-07-20 | 1999-06-25 | Flow cytometer droplet break-off location adjustment mechanism |
Country Status (5)
Country | Link |
---|---|
US (1) | US6079836A (en) |
EP (2) | EP1099105B1 (en) |
JP (1) | JP4426106B2 (en) |
DE (1) | DE69929792T2 (en) |
WO (1) | WO2000005566A1 (en) |
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Also Published As
Publication number | Publication date |
---|---|
DE69929792D1 (en) | 2006-04-20 |
JP4426106B2 (en) | 2010-03-03 |
EP1099105A1 (en) | 2001-05-16 |
DE69929792T2 (en) | 2006-10-19 |
EP1099105B1 (en) | 2006-02-08 |
EP1602915A1 (en) | 2005-12-07 |
JP2002521658A (en) | 2002-07-16 |
US6079836A (en) | 2000-06-27 |
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