IEEE Transactions on Nuclear Science, Vol. NS-30, No. 1, February 1983

LASER-BASED MULTIPARAMETER SYSTEMFOR AUTOMATIC SORTING OF MICROSCOPIC PARTICLES

Bojan Turko and Branko LeskovarLawrence Berkeley Laboratory

University of CaliforniaBerkeley, California 94720 USA

Abstract

The design, characteristics and experimental re-sults of a Laser-Based Multiparameter system for auto-matically sorting microscopic particles are described.The system is used mostly for the analysis of biologi-cal cells which have been stained with fluorescentdyes and suspended in liquids. The cells, passing insingle file through an elliptically focused laser beam,emit fluorescent signals, which are detected by sen-sors and processed, first in analog fashion and thendigitally by a multichannel pulse-height analyzer.The two-color fluorescent and light scatter signalsgive certain parameters of the particles being meas-ured. Sorting rates of several hundred per second areattainable. The high resolution and good stability ofthe system ensure fast, reliable and statisticallymeaningful measurement of particle populations.

Introduction

Numerous microscopic particle separators havebeen described in the Literature (Ref. 1-6). Thesystem described here was mainly designed for themeasurement of physical and biochemical properties ofbiological cells. The cell populations under test arefirst stained with fluorescent dyes. With proper dye-ing and staining procedures, the cells will radiatefluorescence of the desired wavelength when made topass one at a time through an elliptically shaped laserbeam. Since the fluorescent decay time is very short,each cell radiates only during its passage through thebeam. The amount of light radiated is a function ofthe stained volume of the cell. The intensity of theemitted light pulse is a function of the laser beamintensity and shape and also the cell velocity andshape. An optical system of lenses and filters sepa-rates each light pulse into its "red" and "green" com-ponents, focussing each onto the photocathode of theappropriate photomultipl ier.

Current pulses proportional to the incident lightare generated by the photomultipliers. A fast pulsepreamplifier and charge integrator is used in pulseprocessing the signal. The integrator output is avoltage pulse whose amplitude is proportional to thetotal light collected during the passage of the cellthrough the laser beam. The preamplifier output showsthe distribution of fluorescence emitted by a cellwhile it is crossing the beam. Since these signalsare very similar to those found in the detectingcharged particles in experimental nuclear physics, theinstrumentation for analog and digital processing ofthe signals produced by fluorescent cells can be basedon the experience gained in the nuclear field.

The cells cross the laser beam at random. Thesignal shaping and filtering requirements are similarto those in gamma-ray spectroscopy. Linear amplifiersdesigned for the optimum filtering of random pulsesare used in cell signal processing. A multiparameteranalog processing circuit was built for stretchingthe peaks of the shaped pulses along with the coinci-dence logic for the selection of desired categories of

events. Also, analog signals proportional to the quo-tient of pairs of parameters are generated.

The selected signals can be digitized in eitherof two amplitude-to-digital converters and the resultsprocessed in a digital memory. All steps in the ana-log processing can be monitored on an oscilloscope.The digital processing of events can be also viewedwith the aid of the memory monitor display. The re-sults can then be transferred to the external centraldata processor, if first accepted by the operator.

Size of the particles can be determined by mea-suring forward scattered light as each one passesthrough the laser beam. This is done with a photo-diode placed coaxially in the laser beam path to detectthe forward scattered light. A beam dump in front ofthe diode absorbs all of direct light from the laser.An amplifier converts the photodiode current into avoltage pulse which gives an additional parameter forthe analog signal processing.

The above system is made up of a number of opti-cal, mechanical and electrical components. They aredescribed and their function explained in the follow-ing text.

System DescriptionMost of mechanical and optical components of the

system are mounted on a Newport Research Corporationoptical table. This mechanically stable, vibrationfree base improves reliability and performance of thesystem. A diagram of the mechanical system, includingthe optics is shown in Fig. 1. The supporting elec-tronic processing and control components of the system(Fig. 2) are housed in an adjacent rack. Modularcomponents that do not have their own power suppliesare housed in and powered by standard Nuclear Instru-ment Module (NIM) bins.

The light source for illuminating the stainedcells in the flow chamber (Fig. 1) is a Spectra PhysicsModel 171-0 Ion laser with total output power of 10 W.The components of the laser system are the tunablelaser head, a Model 270 Exciter, and a separate powermeter. The strongest lines have wavelengths of514.5 nm (3.7 W) and 488.0 (3.2 W). By employing UVadditional optics, the laser can generate lines of335.8 nm and 351.4 nm (0.07 W each).

The circular laser beam (1.6 mm diameter at l/epoints) is modified by beam shaping optics, consistingof two cylindrical lenses of 20 cm and 2 cm focal len-gth, respectively. The beam passes through two windowsand the center of the flow chamber. A beam dump infront of the exit window absorbs the emerging directbeam energy.

The stained cells cross the laser beam at thecenter of the flow chamber. The beam at this pointhas been shaped into an ellipse by the cylindricallenses. The major axis is about 100 pm and the minoraxis (oriented parallel with the cell flow) less than10 um. By adjusting the 20 mm lens the minor axis can

0018-9499/83/0200-0605$01.00 1983 IEEE

LBL-7520

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be focused at the point through which the stained cellspass. This minimum is about 7 gim wide, smaller than atypical cell diameter.

The flourescence emitted by excited cells ispicked up at the two windows normal to the beam path.A microscope used for system alignment is placed infront of one window and the optical receiver at theother. A filter between the microscope and the flowchamber protects the viewer from scattered light.Water bubbles in the flow chamber might deflect wholebeam into the microscope and cause eye damage if thisfilter were not used.

An Ff1.6 lens at the optical receiver collectsthe fluorescent signal from the flow chamber. Behindthe lens is a barrier filter which blocks the scatteredlight; this is followed by a dichroic interferencefilter which separates the fluorescence light into twocolors. The red and green colors of the fluorescencelight are thus separated and can be detected by twophotomultipl iers.

The shorter wavelength component passes throughthe dichroic mirror and a green barrier filter, and isfocused by another F/1.6 projection lens onto a 260 ,mpinhole. A collimating lens projects the light passingthe pinhole onto the photocathode of the "green" photo-multiplier. The red component of the fluorescence isreflected by the dichroic mirror and by a front surfacemirror onto a third F/1.6 projection lens, which focus-es the red light on another 260 ,m pinhole. A red fil-ter barrier ahead of the lens rejects shorter wave-lengths, preventing any pick-up of the green compon-ents. In the compartment on the other side of eachpinhole a collimating lens projects the light onto aphotomultiplier. Both photomultipliers are RCA Model4526.

A calibrating incandescent light is located ineach photomultiplier compartment. The photomultiplierhigh voltage is turned off automatically when calibra-ting lights are turned on. The lights are broughtdirectly in front of the pinholes when the calibratinglever is pressed. Images of the two illuminated pin-holes, one red and the other green, are projected onthe point in the flow chamber, where the fluorescingcells cross the laser beam. The micrometer whichmoves the surface mirrors should be adjusted until thetwo pinhole images intersect the moving stream offluorescing cells. The laser beam, crossing the imagehorizontally, is also visible due to the Raman scat-tering in water. The point of proper alignment can beseen through the microscope, which can be swung intoposition in front of the viewing window in the flowchamber. In operation, the calibrating lights shouldbe moved back to their rest position so as not toobstruct the optical paths between the pinholes andthe photomultipliers.

The waveform of the output current at the collec-tor electrode of the photomultiplier depends on thebeam cross section and intensity, the shape of fluo-rescing cell and on its velocity. In a typical exam-ple, cells about 10 pm in diameter produce symmetricalpulses of 4 us duration at half maximum (indicating avelocity of 2.5 m/s at the beam crossing).

Each photomultiplier output can be switched eitherto fast current-to voltage preamplifier, or to a chargeintegrating preamplifier (Fig. 1). Viewed on an oscil-loscope, the output of the fast preamplifier shows thefine structure of the fluorescing part of the cell asit moves across the laser beam. The output of theintegrating preamplifier shows a smooth pulse whosepeak value is proportional to the total amount of light

emitted during the cell passage, i .e. proportional tothe stained portion of its volume. ( A circuit diagramof the preamplifiers is shown in Fig. 3.)

The optical receiver assembly is mounted so asto be aligned with the flow chamber. The optimumposition for the output can be found by an alignmentprocedure. Both the flow chamber and the opticalreceiver (except for the fast preamplifiers) weresupplied by the Research Development Company, LosAlamos, New Mexico.

A steady stream of stained cells moves upwards,supported by a liquid in laminary flow sheath (dis-tilled water free of air bubbles) in the flow chamber.The transport of the liquid sheath from the sheathreservoir to the waste reservoir through the flowchamber is controlled by a closely regulated vacuumsystem (Fig. 1). The system consists of a vacumm pumpand a pump motor controlled by a vacuum regulatinggauge. The stained cells, diluted in the samplecuvette, are brought to the flow chamber through athin plastic tube and injected through a 100 gm ori-fice into the center of the liquid sheath. The diame-ter of the sample stream decreases as the cells moveup through the flow chamber, forcing them into singlefile by the time they cross the beam. The adjustmentof vacuum is critical since the slightest turbulencecauses variations in the signal at the optical re-ceiver, decreasing overall resolution of the measure-ment.

The mechanical flow control can change the sheathflow into a turbulent flow that effectively flushesout the flow chamber, eliminating accumulations ofdebris and air bubbles. Fluorescing debris suspendedin sheath liquid can affect the measurement by increas-ing background signal in the photomultiplier. Air pock-ets may get stuck on the walls or windows of the flowchamber and cause laser beam defocusing or even atotal deflection from its proper path.

The Analog Signal ProcessingThe analog processor, including four identical

linear pulse peak stretcher channels with theirassociated logic, is shown in the block diagramsFigs. 4, 5 and 6. A positive voltage pulse at theinput Il is transmitted to a discriminator LD1(LOWER DISCRIMINATOR) and a linear gate LG1. Depend-ing on the mode of operation selected, LG1 will openat the appropriate time to pass the signal unchangedto the peak detector PD1. There the highest pointof the signal is stretched and is available at theoutput for the duration of the HOLD signal, defined bythe logic. When the HOLD signal ceases, the stretcheris quickly discharged and initial conditions restored.

Each analog channel can operate either in an auto-matic mode or in a strobed (gated) mode. Also, anychannel can serve as a master channel to the otherchannels and define coincident events in multiparametersignal processing. Three typical cases will be dis-cussed for which the timing diagrams (not in scale)are shown in Figs. 7 to 9.

Single Channel Mode

Assume that only the first analog channel is used.A signal amplitude exceeding the threshold set by apotentiometer trips the lower discriminator LD1.The Mode switch, which is assumed to be set in AUTOposition, brings the logic "1" level to the gate GAl.It is also assumed that a previous signal is not beingprocessed at the time. The INHIBIT is in logic "O"

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state, and because of the logic inversion at the input,GAl, output is "1", enabling the gate GC1 after pass-ing the logic OR1 circuit. As soon as the lower dis-criminator (LD1) fires, GC1 passes the logic level andsets the latch L1, which in turn enables the peakdetector PDI through the HOLD input. PD stretches and"memorizes" the peak of the input pulse and presentsit to the output terminals.

The signal from Li passes OR21 (whose other inputis set to zero by the mode switch in "IN" position) andcoincidence circuit C, triggering SSA, the first ina series of single shot multivibrators (SSA to SSG)*The coincidence C was enabled because the three re-maining inputs have been set to the logic "1" state.(It is assumed that the mode switches in the three un-used channels were set to "OUT" position, passing thelogic "1" signals through OR22, OR23 and OR24 to C).

SSA through SSG (Fig. 4) generates a sequence ofpulses used for both internal and external strobing,and provide also the inhibit and clear signals con-trolling the four channels. The sequence is as fol-lows:

SSA provides a continuously adjustable delay,followed by three fixed strobe pulses (SSB, SSD andSSF) which are spaced by the intervals defined by SSCand SSE* SSD and SSF outputs are available as EARLYOUT and LATE OUT strobe signals on the front panel.They can be used, for instance, as coincidence signalfor strobing the stretched linear signal into a multi-channel Analog to Digital Converter (ADC). The timingsequence is shown in Fig. 7.

The end of the SSF pulse triggers, through ORp,the dead time single shot SSG. Dead times can beselected by a switch. The sum of SSG and SSH (throughORC) generates a CLEAR signal to all latches and thusremoves the HOLD signal from PD, causing a rapid dis-charge ofthe stretched pulse. The sum of SSC to SSGalso provides an inhibit signal, that through GAl andOR1 closes GCI and prevents acceptance of new eventsuntil the deadtime is over.

Coincidence Mode

Up to four linear channels can work simultane-ously, constituting a four-parameter system for theselection of coincident events. The timing diagramin Fig. 8 shows a two-channel coincidence. The modeswitches in the two unused channels should be in"GATED" and "OUT" positions respectively. In the twooperating channels the mode switches should be set to"AUTO" and "IN" positions.

In either channel an input signal that exceedsthe preset threshold level trips the lower discrimina-tor and by opening the linear gate and setting thelatch, stores the information in the peak detector asdescribed earlier.

However, only if both events exceed the respec-tive thresholds, will the latches L1 and L2 be setand start the sequence of pulses in the single shotsSSA to SSG* If the two discriminators do not overlap,the program will not start and "RESET" single shot (SSH)will be triggered via ORR and GR after the fired dis-criminator returns to zero (Fig. 4). SSH will pro-vide a CLEAR pulse through ORC providing a fast dis-charge of the peak detector. Therefore, only a pairof events that occur simultaneously (i.e. whose dis-criminator crossings overlap) will produce a signal.

Gated Mode

Any of the four analog channels can be a masterchannel, operating as described in the Single ChannelMode Section. The mode selector switches of the mas-ter channel should be in "AUTO" and "IN" positions andthose in the other channels in "GATED" and "OUT"positions.

An event exceeding the threshold in the masterchannel will be stored in its peak detector and startthe sequence generator. The variable delay should beadjusted at the front panel controls so that a DelayedStrobe pulse is generated to be in coincidence withthe peaks of the events entering the remaining chan-nels. Illustrated in Fig. 9 are three possible cases.Only one event (input No. 2) is detected at its peak.The events at the Inputs No. 3 and No. 4 arrived toolate and too early, respectively. This example showsthe importance of making a proper adjustment of thedelay and the strobe width.Single Channel Analyzer Mode (SCA Mode)

Unwanted low-level events in any channel can beeasily eliminated by raising the threshold level. Insome measurements the events exceeding a desired levelshould also be eliminated. This can be accomplishedeither by using an external single channel analyzer(SCA), or using the built-in Upper Discriminator cir-cuit.

External SCA Mode

The output from the selected pulse stretcher chan-nel should be applied to an external Single ChannelAnalyzer (SCA) which will produce a logic output foreach event that falls between the preset upper andlower discriminator levels. This output should beconnected to the SCA IN terminal of the processor(Fig. 5). Provided that the selector is in EXT. posi-tion, the logic signal passes G1 and OR1, setting thelatch Ll, which in turn enables the gate G5. Thisgate is therefore open to pass the Late Strobe signal,which was generated by the processor in the course ofthe time sequence started by the same original event(Fig. 7). The signal is brought out as SCA OUT on thefront panel of the processor and can be used as a gat-ing pulse to the ADC which has the stretched signalof the same event waiting at its imput.

Internal SCA Mode

A built-in upper discriminator eliminates theneed for an external Single Channel Analyzer (SCA).If the selector switch is in INT. position, each RESETpulse from the analog processor will pass G2 and OR1(Fig. 5), setting the latch L1 and thus enable G5.When a Late Strobe pulse is generated (Fig. 7), it willpass G5 and appear as a SCA OUT signal and open thegate to the external ADC. If the output of the se-lected linear channel is connected to the Upper Dis-criminator (UD) input, an event exceeding the presetthreshold level will pass the logic signal from itsoutput through G3 and OR2 and reset the latch L1closing G5. Thus the gate Gs will be already closedby the time the Late Strobe pulse arrives preventingSCA OUT signal from starting a conversion in the ADC.Analog Divider

An independent analog divider has been includedin the Analog Processor. Any peak detector outputcan be used as either numerator (YIN) or denominator(XIN) input for the Analog Divider (Fig. 6). Theoutput of the divider is a pulse whose amplitude

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equals K (YIN/XIN). K is a constant depending onwhich position is selected on gain selector switchnamely, X1,X2 or X5. The linear quotient output canbe digitized in the same way as the signal from thelinear peak detector channels. The early Strobe Out-put of the Analog Processor should be used for thegating of the ADC by the same gating procedure des-cribed under Internal SCA Mode.

Linear Gate

In order to make the system more versatile, alinear gate is included in the analog processor(Fig. 4). An analog signal will pass from the inputto the output of the gate only when enabled by eitherearly or late, internally generated strobe pulse. Anyevent, that is coincident with the operation of theanalog channels of the processor, is thus allowed toappear at the output of the gate.

Circuit diagrams of the analog processor are shownin Figs. 10 through 14. The experimenter can choosewhich one of the numerous'combinations of parametersto use. A typical setting is for the two-color fluo-rometry of the stained cells, shown in Fig. 15. Eachcolor is digitized separately. The selection of livedisplays (normal, contour and isometric) makes iteasier to interpret the results.

A Tektronix RM 15 oscilloscope and an ORTEC, Inc.Model 449-2 LOG/LIN Ratemeter are included in the sys-tem (Fig. 2). The' amplifier and the Analog Processoroutputs can be checked and continually monitored bythe oscilloscope, and the ratemeter measures eventrates at the selected output. Also, two high voltageregulated power supplies for the photomultiplier, andthe pinhole light power source are housed in the samerack, as the power switches, the vacuum gauge and thecomputer interface modules.

Processing of Digitized Events

Two 1024-channel amplitude-to-digital converters(Northern Mod. NS-622) and a 4094-channel digital ana-lyzer (Northern Mod. NS-636) offer sufficient resolu-tion and data storage capacity for most purposes(Fig. 2). Often much less resolution is quite accept-able in sorting out the stained cell populations. Thememory can be divided in 65 separately addresssablesubgroups of 64 channels each. Therefore, a number ofsamples can be'quickly analyzed and compared on theinstant playback display. Either ADC has access tothe analyzer through a data selector, which also pro-vides simultaneous access to both ADC's for two-dimen-sional data analysis (64 x 64 channels). Three displaymodes can be selected for better viewing. The twoADC's provide X and Y address and the number of eventscan be recorded along the Z-axis.

Transfer of data from the digital analyzer to aremote data processor is done in two steps. The firstis from a NS-636 to a LBL builtl-2 Interface Modulefollowed by a Data Transmission System Users TerminalModule (Fig. 2). Both modules were'provided by theLBL Chemical Biodynamics Division.

Experimental Results

The system was primarily designed for flow cyto-fluorometry studies at the Lawrence Berkeley LaboratoryChemical Biodynamics Division. The instrument has beenused continuously for a period of several years. Theperformance of this system and its extensive applica-tion in the cellular biology experiments has been des-cribed in a number of published works (Ref. 7-21). Atypical example is shown in Fig. 16 where changes in a

cell population over a period of several days are shownas a series of spectra. A two-dimensional presenta-tion of another cell population in the Fig. 17 showsthe end result of the two-color microfluorometry. Twochannels of the system's analog processor in coinci-dence mode were used for the selection of the events.X and Y axis represent the intensity of each parameterand the vertical axis the number of events.

Appendix

Principal parts of the systems and their charac-teristics are listed below:

Light Source

A Spectra Physics Mod. 171-0 Ion Laser with Mod.270 power Supply and Mod. 170 Power Meter is used.With the standard optics the laser can deliver a totalof 10 W. The tunable wavelengths (nm) and correspon-ding power (W) are: 514.5 (3.7); 501.7 (0.5); 496.5(1.0); 488.0 (3.2); 476.5 (1.2) and 457.9 (0.5). Ad-ditional lines are available with U.V. optic attached:363.8 (0.07) and 351.1 (0.07).The Flow Chamber and Control Box

They were made by Research Developments Inc., LosAlamos, New Mexico as described in Ref. 2.

Beam Shaping Optics

Two quartz cylindrical lenses (20 cm and 2 cmfocal lengths respectively), each mounted on a NewportCorporation micromanipulator consisting of a tilttable, support post and translation stage.

Two Color Optical Receiver

They were 'also made by Research DevelopmentsInc., Los Alamos, New Mexico. Principal components ofthe receiver are a F/1.6 collimating lens, a barrierfilter, a dichroic mirror, "green" and "red" barrierfilters, and a 260 pm pinhole and a Ff1.6 collimatinglens for each optical channel. Two RCA 4526 photo-multipliers are used for fluorescence detection. Thereceivers each contain a fast preamplifier and an in-tegrating preamplifier (Fig. 3).Vacuum Pump

A Cole-Parmer Instrument Co., (Chitago) Masterflexpump, motor and controller is used. The controller isslightly modified for remote control by the pressuregauge.

Vacuum Regulator

Dwyer Instruments, Inc., Michigan City, IndianaPhotohelic Pressure Switch/Gauge is located in theadjacent rack. The vacuum sensing tube connects thegauge with the waste reservoir. The pressure range isup to 20 psig. Proper pressure for turbulence freeoperation of the flow chamber has to be found experi-mentally.

Optical TableThe laser, the beam focusing system, the flow

chamber and the optical receiver are mounted on aNewport Corporation (Fountain Vallejo, Calif.), opti-cal table. Stable, reproducible operation of thesystem, free of shocks and noise typical in most en-vironments is obtained.

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Ampl if iers

Two Ortec Model 450 Research Amplifiers are used,one in each channel of the optical receiver. The gainof each amplifier can be varied from 2.5 through 3000.Independent active low-pass and high-pass filteringcan be adjusted by selecting time constants from 0.1to 10 ps both for integrate and differentiate modes.High Voltage Power Supply

Two Ortec Model 456 High Voltage Power Suppliesprovide regulated high voltage for the photomultipliersin the optical receiver. They are housed in an ACpowered NIM bin . A High Voltage Time Control Switch(Fig. 2) makes sure that the high voltage is discon-nected when pinhole light for the alignment of theoptical receiver is turned on. Damage of the photo-multipliers may result if the photomultipliers areexposed to too much light when the high voltage isapplied.

Ratemeter

An Ortec Model 449 Ratemeter is used for monitor-ing the event rates appearing at the outputs of theanalog processor. Change of the event rates can beviewed on the ratemeter's dial. An audible sound pro-portional to the rate is also generated as a warningsignal to the experimenter.Four Parameter Analog Processor

This unit was built by the LBL Electronics Re-search and Development Group and is described in theAnalog Signal Processing section of this text.Analog-to Digital Converter

Two identical Northern Scientific Model NS-622Analog-To-Digital Converters are used for the simul-taneous conversion of any two parameters. Maximumnumber of channels is 1024. The resolution can bereduced by a five step switch down to 64 channels.Memory Unit

A Northern Scientific Model 636 Memory System isused for on-line data processing and storage. The4092 channel memory can be divided into up to 64 sub-groups that are individually accessible. Each memorysection can store an individual spectrum. Two-dimen-sional storage and display is possible when the twoADCs are used simultaneously, along with the analogprocessor. Isometric, contour or profile display modecan be selected. A choice of linear or logarithmicdata presentation on the CRT is available. Eitherparallel or serial data transfer to the externalprocessor is possible.Data Interface

The system is used in the LBL Chemical Biodyna-mics Division. Two LBL-built NIM modules are used forinterfacing the Division's internal computer system.The modules are Data Transmission System Users Terminal(LBL No. 16 x 3221) and NS636 to E-2 Interface.

AcknowledgementsThis work was performed as part of the program of

the Electronics Research and Development group of theLawrence Berkeley Laboratory and supported by theNational Cancer Institute and the U.S. Department ofEnergy under contract DE-AC03-76SF00098.

Reference to a company or product name does notimply approval or recommendation of the product by theUniversity of California or the U.S. Department ofEnergy to the exclusion of others that may be suitable.

References

1. Holm, D.M., and Cram, C.S., "An Improved FlowMicrofluorometer for Rapid Measurement of CellFluorescence, Experimental Cell Research 80, pp.105-110, 1972.

2. Steinkamp, J.A., Fulwyler, M.J., Coulter, J.R.,Hiebert, R.D., Horney, J.L., and Mullaney, P.F.,"A New Multiparameter Separator for MicroscopicParticles and Biological Cells", Review ofScientific Instruments, Vol, 44, No. 9, pp.1301-1310, September, 1973.

3. Van Dilla, M.A., Steinmetz, L.L., Davis, D.T.Colvert, R.N. and Grey, J.W., "High Speed CellAnalysis and Sorting in the Flow Systems: Bio-logical Applications and New Approaches", IEEETrans. on Nucl. Sci. NS-21, No. 1, pp. 714-720,February, 1974.

4. Kim, M., Khosrow Bahrami, and Kwang B. Woo, "ADiscrete-Time Model for Cell-Age, Size, and DNADistributions of Proliferating Cells, and itsApplication to the Movement of the LabeledCohort", IEEE Trans. on Biomed. Engineering,BME-21, No. 5, pp. 387-398, September, 1974.

5. Steinkamp, J.A., Hansen, K.M., Crissman, H.A.,"Flow, Microfluorometric and Light-Scatter Mea-surement of Nuclear and Cytoplasmic Size in Mam-mal Cells", The Journal of Histochemistry andCytochemistry, Vol. 24, No. 1, pp. 292-297, 1976.

6. Sharpless, T.K., and Melamed, M.R., "Estimationof Cell Size From Pulse Shape in Flow Cytofluoro-metry", The Journal of Histochemistry and Cyto-chemistry, Vol. 24, pp. 257-264, No. 1, 1976.

7. Bartholomew, J.C., Yokota, H., and Ross, P.,"Effects of Serum on the Growth of Balb 3T3 A31Mouse Fibroblast and an SV40-Transformed Deri-vative", J. Cell Physiol. Vol. 88, pp. 277-286,1976.

8. Bartholomew, J.C., Neff, N.T., and Ross, P.,"Stimulation of WI-38 Cell Cycle Transit: Effectof Serum Concentration and Cell Density", J. CellPhysiol. Vol. 89, pp. 251-258, 1976.

9. Teng, N., Bartholomew, J.C., and Bissell, M.J.,"Insulin Effect on the Cell Cycle: Analysis ofthe Kinetics of Growth Parameters in ConfluentChick Cells", Proc. Natl. Acad. Sci., Vol. 73,pp. 3173-3177, 1976.

610

10. Hawkes, S.P. and Bartholomew, J.C., "QuantitativeDetermination of Transformed Cells in a MixedPopulation by Simultaneous Fluorescence Analysisof Cell Surface and DNA in Individual Cells",Proc. Natl. Acad. Sci., Vol. 74, pp. 1626-1630,1977.

11. Teng, M.H., Bartholomew, J.C., and Bissell, M.J.,"Synergism Between Anti-Microtubule Agents andGrowth Stimulants in Enhancement of Cell CycleTraverse", Nature, 268, pp. 793-841, 1977.

12. Wade, C.G., Baker, D.E., and Bartholomew, J.C.,"Selective Fluorescence Quenching of Benzo(a) pyrene and a Mutagenic Diol Expoxide in MouseCells", Biochem., 17, pp. 4332-4337, 1978.

13. Becker, J.F., and Bartholomew, J.C., "ArylHydrocarbon Hydroxlylase Induction in Mouse LiverCells in Culture", Chemico-Biol. Inter., 26, pp.257-266, 1979.

14. Wade, C.G., Rhyne, R.H., Woodruff, W.H., Bloch,D.P., and Bartholomew, J.C., "Spectra of Cells inFlow Cytometry Using a Vidcon Detector", J.Histochem. and Cytochem., 27, pp. 1049-1052, 1979.

15. Bartholomew, J.C., Pearlman, A.L., Landolph,J.R., and Straub, K., "Modulation of the CellCycle of Cultured Mouse Liver Cells by Benzo(a)pyrene and Derivatives", Cancer Res., 29, pp.2538-2543, 1979.

16. Greasey, A.A., Bartholomew, J.C., and Merigan,T.C., "Role of GoGl Arrest in the Inhibition ofTumor Cell Growth by Interferon", Proc. Natl.Acad. Sci., 77, pp. 1471-1475, 1980.

17. Peterson, J.A., Bartholomew, J.C., Stampfer, M.,and Ceriani, R., "Analysis of Expression of HumanMammary Epithelial Antigens in Normal and Malig-nant Breast Cells at the Single Cell Level byFlow Cytofluorimetry", Exptl. Cell Biol., 49,1-14, 1981.

18. Parry, G., Bartholomew, J.C., and Bissell, M.J.,"Growth Regulation in RSV Infected Chicken EmbryoFibroblasts: The Role of the Src", Gene Nature288, 720-722, 1980.

19. Bartholomew, J.C., Hughes, A., and Das, K.,"Alterations in DNA Synthesis in Mouse LiverCells Caused by the Tumor Promoter 12-0-Tetradecanoylphorbol-13-Acetate (TPA) ", J. Supra-molecular Structure. In press.

20. Cohen, S.R., Burkholder, D.E., Varga, J.M.,Carter, M., and Bartholomew, J.C., "Cell CycleAnalysis of Cultured Mammalian Cells After Expo-sure to 4, 5. 8-Trimethylpsoralen and Long-WaveUltraviolet Light", J. of Investigative Dermato-logy, 76, pp. 409-413, 1981.

21. Greasey, A.A.T.C., "DirectAction in thepress.

Bartholomew, J.C., and Merigan,Evidence for Interferon's Site ofCell Cycle", Exptl. Cell Res. In

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GREEN BARRIER FILTER RED BARRIER FILTERSPECTRA PHYSICS DCRI IRR ,2-,9FRTSRAEMRO

MODEL 270 FIRST SURFACE MIRRORPOWER SUPPLY BARRIER FILTER q-

F1.6 LENS_Cp; ~~~~FLOW CELL _ FLUORRSCENCE IIH CTEFLOWCELL ~SIGNAL

SPECTRA PHYSICS m LIGHT SCATTERMODEL171k PHOTO SIGNALIONLASER

BEAM SHAPINGDID

it ~~~~OPTICS BEAM DIODE BIASi~~~~ - D~~~~UMP

Fig. 1 Laser-based multiparameter system for automatic sorting ofmicroscopic particles: General block diagram.

FROM PMT UK tk mvv *vv("REDMLFE- AMPLIFIERFROM PMT ORTEC MODEL 450

PREAMPLIFIER AMPLIFIER _("GREEN")_

IOX3411P-1

S C ATT ER CURN

ORTEC MODEL 449-2LOG/LIN L_ _

RATEMETER

1OX2 171P-1

TO DATA

PINHOLE-TO PINHOLE IGHTL

LIGHT POWER SUPPLY

TUBE FROM DWYER SERIES 3000VACUUM VESSEL

= PHOTOHELICPRESSURE

_d*~~ SWITCH/GAUGETO PUMP MOTOR

CONTROL

Fig. 2 Electronic component block diagram of the system,

I O.jjF-12V

PREAMPLIFI ERXBL 828-11061

Fig. 3 Optical receiver photomultiplier, fast preamplifierand integrator circuit diagram.

611

XBL 828-11060

0 TO +10VLINEAR GATE PDI

I 3 PULSE INPUT 10 TO *^OV) DICIILO*E RDTCOINHIBIT IHEOLD,SL;_1Q

AUTO I A 1 - L OUT IGATED CLEAR_0 ILN -O O2AUTO

GATED I G1 OR1 LINEAR GATE PD2

Ir j F PUSE IPT TO + E r DSC R MI1NATO DTECOR]1.I INHIBIT 1-02QAUTO

--I 12 OUTGATED 0 CLEAR IN 0-0 0R22

AUTO O-IQ

ATES 1... |G62 ORZ LINEAR GATE PD30

~~~~~~~~~~LWR L3+108)DISCRIMINATORI PU L SE IN"P UT O TO +lIO V) 1HOOLD R [|- * < | ~~~~~~~~~~~~~~LATCH HOLD

F INHIBIT 13DIQAUTO 1 GA3 L3 OUT 1

GATED 0- CLEAR IN O-.Q OR23AUTO0 _

| TO +10V

0 TO +1OViti i - - "-

144

OUTPUT-r3 01A

01 B

OUTPUTiz3 02 A

L* 02 B

OUTPUT03A03B

GATED 1- ) OR3 LINEAR GATE PD4 IIIIOTU

IBITLARE H 304

I Q I HRtSHOLDltFE.rr_ 42, I I I a 04 BGATED 04 LI ILA_ N 0-) OR24 N IIIEICERESETAUTO 0 II1RESET

GATED1IORIORR I SSHISTROBE I

INHIBIT TLATE STROBE SINGLE

,...,.-

DELAYED STROBE =EARLY STROBE

DELAYIORI EARLY LATE TO UPPERDELAY ~~~~~~~STROBE STROBE DISCRIMINATOR

IN ~~LINER OUT SSR S DS SSE SSFIN~~~~GAT SSA

EARLY GATESINGLE SINGLE SINGLE SINGLE SINGLE SINGLESHOT SHOT SHOT SHOT SHOT SHOT

9LATE :ORP

DEADTIME

SSG ORCSINGLESHOT CLEAR

XBL 828-11062

Fig. 4 Four parameter analog processor module block diagram (discriminator, stretchers and logic).

ANALOA PROCEPRSOCR

ISCRMINALATE LATE OUT(+5 VMAX.)EEX.12

RESET FROM INT. I 5-ANALOG PROCESSOET.OR SCA OUT

INT. 1 0 L t

DISCRIMINATORO TO +10V LEVEL

DISCRIMINATOR XBL 828-11063

Fig. 5 Four parameter analog processor moduleblock diagram (upper discriminator).

THRESHOLD;INPUT PULSE 7 YSE SI

LOWER DISCRIMINATOR R1

PEAK DETECTOR(OUTPUT)

-ADELAY (VARIABLE) S5DELAYED STROBE SSB f X

SSC f5IEARLY STROBE SS0

SSE

LATE STROBE SSF

DEAD TIME SSr

RESET

CLEAR

INHIBIT

XBL 828- 11064

SSHFL-

I IXBL 828-11065

Fig. 6 Four parameter analog processor moduleblock diagram (analog divider).

612

I3 g

I4 [

Fig. 7 Single channel mode timing diagram.

i i

0

--m~~

THRESHOLD #1 HINPUT PULSE #2 -

THRESHOLD #2 7= HSEEIINPUT PULSE #2

HYSTRESI

LATCH (HOLD) #1

PEAK DETECTOR #1

PEAK DETECTOR #2(OUTPUT))

DELAYED STROBE SSB

EARLY STROBE SSD 7SSE

LATE STROBE SSF 7

DEAD TIME SsC f

RESET SSII

CLEAR

INHIBITl

XBL 828-11066

THRESHOLD iINPUT PULSE #1 T -

UPPER DISCR #1

PEAK DETECTOR #1_ OUTPUT l

DELAY (VARIABLE) SSAfDELAYED STROBE SSB

EARLY STROBE SSO

SSEwLATE STROBE SSFDEAD TIME SSGRESE _SCLEAR

INHIBIT

INPUT PULSE #2 ;\_STROBEi 1*

PEAK DETECTOR #2OUTPUT

LATCH (HOLD) #2

INPUT PULSE #3

PEAK DETECTOR #3

LATCH (HOLD) #3

Fig. 8 Coincidence mode timing diagram,

/ l ~~60STROBEINPUT PULSE #4

PEAK DETECTOR #4

LATCH (HOLD) #4 f IXBL 828-11067

Fig. 9 Gated mode timing diagram.

IAOUTBOUTCOUTDOUT

Fig. 10 Peak detector and lower discriminator circuit diagram.

613

5TROBE-W/.--ISTOB -1

& 'L-I -1--

614

3 2 S PA C

6 5V +5VYPH

M S -011 35. SCALACHG4A0T7E0OUTTO+5vMM2

748-00 ATOL 828-11069O

+ 5 V M2O121 1O22652TOM2

+59~~~~~~~~~~~5 2 5

FROM M21~~~~~~SMTOM2

1 NSSG

~~~~z ~MIO234+59 0K. 0+v 2

+5++9 +5 + +5V I + M2 +59

M2 EL8OUT SCG6+ln E GA ED 20.TO2EM24T 2505 5 ELPO M25T

50.10 1%1%1% 1% 1%~~~~~~~~~~~~~~~~~~~+V2 M23TO5V7

67 54 15 14 156 7 14 15 B i~~~~~~~~~~~~~~~~~4 14- 15 6M20-M4,31s 1- '~~~~~'T p513 BjQ5 1j5 Q13~~~~~~AUTI9TZ S K 742SM2 4L2

M28 M2B M2BM29 M31 M3~ ~ ~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~12 32 M3FROMlS55~~~~~~3l2 2g1~~~~~l4 2~~5V 4 i , l FRO M30-12 0M241N441 1( 3 31 31

SCAlKu 1O1444K1944 DN447 1 A447 ED9447STREATBEE

1N4447 130pF

FROM MIO-5 tF 5.1V |DADIE|gpFFROM M15-5 , .FROM M20-5 ?()tt

1N4447xI4 TIIT I I T1. w Z 4> Lz Z Z ZWm

-0 0 -0 -0 0An 0 0_ 9 82 117

~n C XBL 828-11070

Fig. 12 Time sequence circuit diagram.

615

XBL 828-11071

Fig. 13 Analog divider and linear gate circuit diagram.

X100X50

X25

X10.0X5.0

X2.5

x1.0XO.5

- 23 6 O OUTPUTG A 5OOi~~~~~~~~ AD507 6SGD-IOOA

EG & G3

a-12 V 1pF~~~lp510o p

O.lyuF 4.7,mF-1V +21 +I130VXBL 828-11072

Fig. 14 Light scatter photodiode current amplifier.

616

-

I J . . . . . -----.I BEAMSHAPER

REGULATEDNI SNHF NI IHIGH VOLTAGE OSCILLOSCOPEr+ ~~~SU PP N Yi |e OWERSUPPLNYLPPL POWER SUPPLY

POWER SUPPLY#2

DAY I DAY 2 Dtl9 DAY3 DMSO _DAY4DMSO DAY2 BaP

DAY3 BaP DAY4 BaP DAY2 DE DAY3 DE DAY4 DE

DNA CONTENT (ARBITRARY UNITS)XBL 7811-13050

Fig. 16 Spectra of the changes in a cellpopulation for a period of severaldays (Courtesy of J. Bartholomew,LBL Chemical Biodynamics Division)

XBL 828-11073

Fig. 15 Typical system set-up for two-color flow microfluorometry.

NS 3735 1 FERRITIN FITC BINDING TO SERUM CELLS

A

0.0

44.

6000

5250.0 tO

4500.0 t

3750.0 o

3000.0

2250.0

*so0.0

o.o

16.0,..0-' Ges320

60o

XBL 828-11074

Fig. 17 Two-dimensional distribution of acell population. Two-color opticalreceiver and the analog processor incoincidence mode was used (Courtesyof J. Bartholomew, LBL ChemicalBiodynamics Division)

NMuLi CI8

0,05

0.04

0.03

z0

a.0ILw

UI.0z0P10

0.02

0.01

0,04

0.03

0.02

0.01

..0

6.