Two-parameter data acquisition system for rapid ... - Wiley Online Library

Cytometry 8:83-90 (1987)

0 1987 Alan R. Liss, Inc.

Two-Parameter Data Acquisition System for Rapid Slit-Scan Analysis of Mammalian Chromosomes' H. -U1. Weier and W.G. Eisert AG Zytometrie, Gesellschaft fiir Strahlen- und Umweltforschung Munchen, D-3000 Hannover 21, Federal Republic of Germany Received for publication June 2, 1986; accepted August 13,1986

A data acquisition system is described for recording two independent signals simultaneously from a laser-based flow cytometer for rapid slit-scan chromosome analysis. Highaperture microscope optics allow recording of fluorescence distributions along the longest axis of metaphase chromosomes with a spatial resolution better than 1pm. Fluorescence and small angle forward light scatter as well as dual-wavelength fluorescence signals from Indian muntjac chromosomes stained with propidium iodide (PI) or acridine orange (AO) have been recorded simultaneously. While maintaining the multi-user operation of the computer, photomultiplier signals are digitized at a rate of 400 signals per second,

Flow cytometric investigations show low spatial resolution in most cases. However, various intracellular materials and coarse morphological structures have been measured by scanning along the cell axis (26). By measuring the total amount of one or more dyes bound to individual chromosomes, it has been shown that the human karyotype may be resolved into 20 groups showing distinct peaks with low spatial resolution (15,18). Slit-scan flow cytometry provides higher spatial resolution and has proved to be a valuable tool in the analysis of cells and cell constituents (2,3,9,11,12).In addition to the total amount of DNA per chromosome, the corresponding centromeric index has been measured (11,17). Simultaneous scanning of additional parameters such as forward or 90" light scatter or fluorescence in a second wavelength interval offers the possibility of discriminating further structural differences along the chromosome as well as detecting preparation artifacts (20). In the past, slit-scan analysis signals have been recorded using single-channel transient digitizers (9,10,11,12)or sophisticated analog signal processing circuits (26). With these techniques it has not been possible so far t o record two independent signals simultaneously with high resolution. Alternatively, two-parameter in-

stored temporarily in high-speed cache memories, and transferred subsequently to a minicomputer for further storage. Extensive software packages for data acquisition, analysis, and display of the results are described. Data acquisition is generally done in list mode, allowing complete reconstruction of individual signals (profiles) at any time. The distribution of stained constituents along the chromosomes can be displayed. Furthermore, histograms of various parameters of the input signals may be generated. Key terms: Flow cytometry, slit-scan chromosome analysis, dual-channel high-speed transient digitizer, data acquisition

formation has been recorded as sequential data files in a single-channel waveform digitizer by using two different analog amplifiers, with one signal being delayed for a sufficient time (14).In this sequential procedure the temporal coincidence of the two input signals is lost. To prevent overlap, a sufficient delay between both signals has to be selected. The amount of data to be transferred, including the data representing the delay period, is significantly increased. This leads t o greatly reduced overall data acquisition rates. Commercially available transient digitizers are often equipped with a standard interface (IEEE 488, RS 232, etc.), which further limits the data transfer speed. Until recently fast analog-to-digitalconverters (ADCs) were too expensive for the average research laboratory. Therefore it had been advisable to store the analog signal in real time by using analog registers and to digitize at low speed (5,161. For multiparameter analysis at moderate speed, a system containing a number of multiplexed analog input channels but only one fast ADC Address reprint requests to H.-UI. Weier, gsf Miinchen, Herrenhauser StraBe 2, D-3000 Hannover 21, West Germany. 'Portions of this work were presented at Analytical Cytology XI, Hilton Head, South Carolina, November, 1985.

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WEIER AND EISERT

seems to be a n acceptable solution (13).As monolithic video digitizers are now offered at moderate prices, fast analog-to-digital conversion is available for the average laboratory. Those flash converters with aperture times of some nanoseconds do not need additional sample-andhold circuitry and therefore simplify system development considerably. In this paper we introduce a data recording and analyzing system for slit-scan flow cytometry that is capable of digitizing and storing two-parameter input signals simultaneously. High-speed data acquisition can be provided without compromising accuracy of the measurement. Our system consists of homemade hardware for signal processing, digitization, and temporary storage of data; a parallel interface to a commercially available minicomputer; and extensive software for data acquisition, correction, and calculation. Software for histogram processing, display of results, or for statistical calculations may be taken from existing packages (1,19) or the SCAN library (25). The program library may easily be extended for different or altered calculations on diskfile data. Although application of this instrument is not limited to chromosome analysis, our interest so far has been focused on the detection of alterations of chromosome structure owing to thermal or chemical treatment.

DESCRIPTION OF THE SYSTEM Hardware The hydrodynamic system a s well as the major optical parts have been described in detail previously (6,7). The hardware composition of the entire system for highspeed slit-scan analysis is shown in Figure 1. The flow cytometer (laser spot diameters 0.8 x 42 pm) is connected to the waveform recorder, which continuously digitizes the analog input voltage and stores the information as 64 points of 8 bit data in a high-speed memory. The sample rate may be chosen between 0.23 and 20 megasamples per second to accommodate different flow velocities. Each signal data is transferred to the minicomputer (Eclipse Si140, Data General Corp. [DG], Westboro, MA) and stored on disk (Trident T302, Century Data Systems [CDS], Anaheim, CA). While one terminal serves as a n interface between the user and the operating system (DG/AOS, Advanced Operating System) a second, graphic terminal (TEKTRONIX 4051, Tektronix, Beaverton) is used to display calculated histograms in realtime. A hardcopy unit may be attached to the graphic terminal to document the slit-scan profiles or histograms. A Winchester disk drive (DG 6046, capacity 12.5 Mbyte) stores the operating system software and data acquisition programs. Under the limitations of the operating system and a multi-user/multiprocess environment, data acquisition rates do not exceed 400 event&. Although disk space up to 600 Mbyte is available for storage of list mode data or histograms, a magnetic tape drive (DG 6026) is routinely used for data backup.

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FIG.1. Block diagram of the two-parameter data acquisition system. The transient recorder digitizes the photomultiplier signals at high rates and stores the data. The data is then transferred to a minicomputer and stored on disk. One terminal is used to initialize and stop the data acquisition, while resulting histograms may be displayed on a graphic terminal (TEKTRONIX).

Transient digitizer. A block diagram of the transient digitizer is shown in Figure 2. Preamplifiers. The photomultiplier signals are fed into separate preamplifiers providing a gain factor of 20, Monolithic, high-speed operational amplifiers (1321/ 1322, Teledyne Philbrick, Dedham, MA) are used to realize slew rates as high as 35 V / p . The photomultiplier voltages are adjusted between 400 and 700 V to get preamplifier output signals of 2 V maximum. To satisfy the special needs of the ADCs the signals are further amplified by a gain factor of 5 and inverted using high-speed, field effect operational amplifiers (LF 357, Siemens, Furth, FRG). A positive, 5-V trigger signal is generated by a voltage comparator (SN 72710, Texas Instruments, Freising, FRG). The trigger level may be adjusted manually. Analog-to-digital converters and high-speed memories. Two 6-bit flash converters (CA3300, RCA, Somerville), selected for speed and linearity, are cascaded to deliver a resolution of 7 bit + overflow bit a t a maximum sampling frequency of 20 MHz. The memories for temporary storage are mounted in close proximity to the ADCs and are organized a s two times 64 ~9 bits (82919, Valvo, Hamburg, FRG). The parallel data is latched by the memory in 35 ns per word. The analog input is sampled continuously to minimize system latency times and to enable recording of data prior to the trigger. Following the trigger pulse, a limited number of points are recorded, a computer interrupt request is generated, and further digitization is disabled. Then the transient digitizer is turned into readout mode. Computer interface. To maintain maximum transfer rates, data from both channels are always transferred in parallel form. A digital LtO-interface as well as a general purpose interface with DMA option may be used on the DG computer. The interface of the waveform digitizer contains buffers for all data lines and a number of monostable multivibrators to provide fixed pulse lengths for handshake signals. All data and control lines contain high-speed optocouplers (IL102, Siemens, Fiirth,

85

TWO-PARAMETER DATA ACQUISITION SYSTEM

1 I

5 .sl

TRIGGER

I

MEMORY

w

0

2 K

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LOGIC RESET

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FIG 2. Block diagram of the modular transient digitizer. Flash converters and high-speed cache memories allow up to 20 conversions per microsecond. The analog signal processing, a parallel computer interface, a test generator, the control and timing logic, and the power supply are integrated in one cabinet (OF = overflow bit).

FRG) to separate the different circuit potentials. The computer output lines are used as control signals. They increment the word pointer in the digitizer memory and reset the system. Test pulse generator. A pulse generator simulating slit-scan chromosome pulses is integrated in the transient recorder. The usefulness of the generator for hardware checks and software developments has been reported in detail previously (24).

Software Program initialization. Data acquisition and processing are started by a system macro call. The schematic (Fig'3)lists the programs contained in the library SCAN (25)' Programs are loaded and executed by menu tions. One task Per console, either data acquisition O r calculation, may be initiated by the user. Because of the fact that AOS allows several tasks to run in parallel, an additional terminal offers the possiblility of beginning data evaluation or reduction procedures immediately after the initiation of data acquisition. Data input. At the time when either a single- or dualparameter program is called, the entry of a maximum runtime for data acquisition is requested. As soon as an interrupt request is acknowledged, all 64 memory locations of the digitizer are read into the minicomputer by the interrupt service routine. The time to transfer 64 words of 16-bitdata amounts to less than 400 ps for both interfaces. Immediately before returning to the main program, the waveform digitizer is reset, which also restarts the continuous digitization of the analog input.

FIG,3, &hematic ofthe SCAN-program library with menu selection of desired programs for data acquisition or correction, calculation, and display of the results. The structure allows further programs to be easily implemented.

Data storage. The acquisition program maintains a data buffer of 4,096 bytes to store data from the I/Ointerface. When buffer space is exhausted, i.e., after acquisition of 32 complete signals, data transfer to the disk is initiated. During this time the operating system AOS ignores all interrupt requests. With a CDS 300Mbyte removable disk drive, the data transfer rate from the computer to the disk is as high as 900 kbytels. Realtime histogram display. Integral values of input signals are calculated simultaneously with acquisition of list mode data. During single-parameter data acquisition, a histogram is accumulated and displayed,

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whereas during dual-parameter data acquisition a “dot- nel. In addition, the corresponding single-parameter plot” is generated in real time on a graphic screen to histograms are calculated and, as described above, norfacilitate fine tuning and monitoring of the system. malized to 512 channels. At program start one filename (NAME) has to be entered for the resulting histograms, Software algorithms which will be stored as B.NAME, l.NAMEX, and Single-parameter data analysis. Data stored in each 1.NAMEY. of the 64 scan points are added to form the pulse inteDual-parameter routines for baseline restoration, noise gral. Baseline restoration or noise subtraction may be subtraction, smoothing, or WINDOW are also available. performed prior to the final analysis by creating new, The two types of existing graphic program packages secondary data files. Histograms for the integral values differ in their application: one is intended to display are calculated, normalized t o 512 channels by division histogram data, the other shows original profiles from by eight, and stored on disk. They may be processed for the list mode data files. These programs are described display as well as for further statistical analysis (Gaus- in detail elsewhere (25). sian fits, smoothing, separation of subgroups) by softEXAMPLES OF APPLICATION CHROMOSOME ware either within the SCAN library or by other PREPARATION AND STAINING software tools. Prior to analysis, list mode data may be processed by Exponentially growing Indian muntjac cells (Flow, WINDOW, discriminating those data sets, in which a Meckenheim, FRG) were blocked for 8-12 h by Colcemid selected parameter is not within a defined window. (0.04 pglml in HAM F-10 supplemented with 15% FKS Detection of centromere. Fluorescence intensity data and 2% antibiotics). Harvesting of cells, swelling, and of each signal are scanned for a “relative minimum.” staining with propidium iodide (PI) was performed acThe “relative minimum” is defined as the point of the cording to the procedure described by van den Engh et signal being surrounded by sections of higher ampli- al. (23). In contrast to the method of van den Engh et al., tude. In order to minimize erroneous detection of noise the samples were irradiated in an ultrasonic bath (Elma minima, a minimum “valley condition” has t o satisfied K42, Singen, FRG) for 30 s to release the chromosomes. at least two or three points on each side of the suspected This turned out to be more gentle than syringing the minimum have to show a steady increase. The program cells through a 22-gauge needle (20,221. To study the reassociation kinetics of chromosomal may be modified to recognize only minima with 4 to 5 or more neighboring points of higher amplitude. This will DNA in situ after thermal denaturation, the procedure result in a search for a deeper, more pronounced mini- outlined by Stockert and Lisanti was used (21). Chromum. The computer algorithm starts from the left, the mosomes were prepared as described above, but without first dataword in the record, searching for a minimum. PI in the swelling b a e r (23). The chromosomal DNA If a minimum is detected, the same procedure will be was denatured by heating at 100°Cfor 3 min in 2.0 X SSC repeated in the opposite direction starting at the (SSC: 0.15 M NaC1, 0.015 M Na3 - citrate) with 10% extreme right point (last in the record) going to the left. formamide (Merck).The reassociation was then allowed If both (left and right) searches find the minimum at the to take place for 2 min, before formaldehyde was added same place, the program is terminated. If right and left to a final concentration of 5%. After 2 min acridine minima are different, it is assumed that the signal shows orange (AO) in 2.0 x SSC was added to a final concentraat least two minima (centromeres). The left position is tion of 3 pglml. stored, and the program continues from the left to the RESULTS right until both search directions have found identical This system is routinely used for slit-scan chromosome positions. Dual-pammeter registration and display. With the epi- analysis in conjunction with our homemade flow system. illumination optical design with dichroic mirrors as de- The sums of the 64 points of each chromosome scan have scribed by Eisert (7), different fluorescence wavelength been used to generate the histogram of the DNA conintervals as well as light scattered in small angle for- tent. A typical single-parameter histogram of the relaward direction are accessible simultaneously. By con- tive amount of DNA of propidium iodide stained Indian vention the lower 8 bits in each 16-bit dataword contain muntjac chromosomes is shown in Figure 4. Peaks cordata recording the double stranded DNA related fluores- responding to the DNA content of chromosome # 1, # 2, cence signal. The profiles of the scatter and fluorescence and # 3 are easily distinguishable. The aggregates of signals may be displayed on the graphic screen either the # 3 and the X-chromosome form a separate peak alone or together with calculated ratios. The program between the # 1 and # 2 chromosomes. In most of the for data evaluation AWERT allows any combination of histograms, Y chromosomes appear as a separate peak. two parameters of pulse-half width, -integral, or -peak The coefficient of variation (C.V.) for measurements of for the upper and lower byte to be calculated. A variable the relative DNA content of #1 chromosomes 1 was scale factor for each parameter permits the distributions found to be between 2.8% and 4.0% in most of the runs. This C.V. turned out to be sufficient to identify all scan to be altered for special applications. The resulting histogram is organized in 64 by 64 chan- signals from # 1 chromosomes and to isolate those pronels, the maximum count is limited to 32,767 per chan- files from the remaining list mode data. After the pri-


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FIG.4. DNA histogram of the chromosomes of the male Indian muntjac (PI staining: total number: 23,000: C.V. of the chromosome #1: 2.8%). Individual peaks are indexed with the chromosome number with respect to their relative DNA content.

mary classification of the chromosomes, the individual profiles were checked for the location of the centromere. The percentage of signals from # 1 chromosomes showing a significant minimum was dependent on the preparation and ranged between 60% and 80%. Dual-parameter measurements have been performed on PI-stained metaphase chromosomes. Low angle forward light scatter signals were recorded simultaneously with the fluorescence signals. For chromosome lengths ranging from 0.3 to 20 pm, the digitization rate was set to ten megasamples per second in order t o capture even the longest signals. This is equal to an observation time interval of 6.4 ps or 32 pm at a flow velocity of 5 d s . Figure 5 shows an example of the fluorescence and scatter intensity along different chromosomes of Indian muntjac cells. The fluorescence as well as the forward light scatter signal shows a significant decrease of the relative intensity in the middle of the first and second signal. Because of their relative total fluorescence intensity these signals were assumed to be related to #1 chromosomes. The signal-to-noise ratio of the forward light scatter signals of Indian muntjac chromosome samples has always been acceptable. A number of light scatter signals of # 1 chromosomes showed a relative minimum coincident with the decrease of fluorescence in the region of the centromere (Fig. 5, first and second signal). Another example illustrates the simultaneous registration of red and green fluorescence intensity within small segments of AO-stained chromosomes (Fig. 6). After thermal denaturation regions of different reassociation velocity of double-stranded DNA can be observed

along metaphase chromosomes (21). The reassociation process is stopped after 3 min by addition of formaldehyde. By recording profiles for the red and green AOfluorescence segments of slow and fast reassociation can be differentiated. Figure 6 shows signals, which were recorded on an AO-stained,partially reassociated Indian muntjac chromosome # 1(Fig. 6, left). For each segment the redigreen ratio is calculated and displayed (Fig. 6, right). Bands reflecting the different reassociation velocities by extreme values of the redlgreen ratio can be localized. Owing to the fact that dual-parameter data from the transient recorder are transferred in parallel as 16-bit words, single- or dual-parameter data acquisition is performed with the same speed. In the examples shown in Figures 5 and 6, an entire signal is recorded in 6.4 ps. Upon interrupt request, the operating system of the Eclipse computer first saves the processor status and registers before serving the interrupt. The transfer of data is then done in another 400 ps. System latency limits the data acquisition by interrupt request to approximately 400 events per second. The system’s data channel facility allows rates as high as 7,000 recorded signals per second to be achieved. The Eclipse computer, under the multiuser/multitasking operating system AOS, allows only simple calculations to be performed in realtime. Realtime histograms of the signal integrals are calculated and displayed on the graphic screen in the form of “dot-plots.”Depending on the number of tasks running in parallel, their individual priority, and the number of data channel transfers such as disk data transfer, up to 200 signals per

WEIER AND EISERT

88

T

FIRST

SECOND

THIRD

TIME

FOURTH SIGNAL

-

FIG.5. Typical profiles for the PI fluorescence and forward light scatter signals as recorded from different Indian muntjac chromosomes. In the first and second profile the centromere may be clearly distinguished as a minimum in the fluorescence signal.

4

1

FIG.6. An example for dual-parameter data acquisition investigating the amount of double versus single-stranded nucleic acids along metaphase chromosomes. Left: The intensity of green (below) and red (above) fluorescence light has simultaneously been recorded along AOstainod (3 pg/ml) chromosomes. Pairs of the original signals as recorded

from the photomultiplier tubes are displayed on the graphic terminal. Right: The ratio of red-to-green fluorescence light has been calculated for each of the 64 points and is plotted as a function of the chromosome locus (x).

second can be processed for realtime histograms calculation and display. Such histograms can be used to check the system performance and to allow a quick estimation of the resulting distributions. Time-consuming programs like dual-parameter histogram calculation of peak, half-width, or integral of pulses are executed immediately after the onset of data acquisition. The detection of minima, the determination of a

centromeric index, or fluorescence-to-scatteredlight ratio along the chromosomes are usually executed after data acquisition is completed. If disk space should be lacking, the multi-tasking operating system offers simultaneous data acquisition and transfer of files on the tape. Typical run times for data analysis programs are listed in Table 1.These routines are programmed using DG’s

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Table 1 Typical Run Times for Frequently Used Data Evaluation Programs One slit-scan profile [ms] Single-parameter calculation Integral Half-width Peak Centromeric index Dual-parameter calculation Integral vs. integral Half-width vs. peak Discrimination of dual parameter data by WINDOW Ratio (signal UsignalZ)

Runtimea One megabyte list mode data [s]

8.9 12.8 8.7 1,030

138.3 200.0 135.1 16,067

17.9 17.5

139.8 136.4

18.1

141.9

3,730

29,140.6

aThe data are fetched from the disk datafile in records of 64 (singleparameter) and 128 (dual-parameter) bytes. The times include generation of histograms and output of results or graphic on the terminal.

With respect to the optical resolution of the flow cytoFORTRAN 5 compiler. A floating point processor will further expedite the execution of compiled FORTRAN meter, the bandwidth of the preamplifiers turned out to be sufficient. The trigger delay caused by the slowprograms. switching 72710 comparator was not critical. By means of the selectable pretrigger, the available memory in the DISCUSSION digitizer may be partitioned. If a high trigger level is The data acquisition system described in this paper required to discriminate background fluorescence, the has been shown to be capable of digitizing, storing, and number of scan points to be stored before the occurrence transferring dual-parameter data such as fluorescence of the trigger signal may be selected to accommodate or light scatter signals a t high rates. In contrast to slow or fast rising signals. commercially available transient digitizers, the parallel At a flow velocity of 5 m l s , the maximum digitization interface for transfer of stored digital information to a rate of 20 sampleslps is equal to a spatial resolution of 4 computer or other mass storage devices allows data pixeldpm, which is far beyond the optical resolution. transfer rates of more than 100,000words per second. The high-speed digitization becomes very useful for flow The Data General multiuserlmultitask operating sys- rates of 10 m l s and more, which are realized in hightem allows repetition rates of up to 400 eventsls with speed analyzers (6). the interrupt request line. The operating system, how- The modular hardware concept of the transient digiever, allows simultaneous calculations or display of the tizer has kept the system highly flexible. With respect results. In direct memory access mode, the input data to the larger chromosomes a n extension of the highrates can be increased up to 7,000 signal scansls without speed memory of the waveform digitizer is advisable. compromising overall writing speed on disk, if 64 data- Owing to their low chromosome number Indian points per individual analog signal are recorded. This muntjac chromosomes provide a convenient model sysrepetition rate will be decreased if signals are recorded tem to test this instrument (3,4,8,27). Single- and dualwith a higher number of data points. parameter data acquisition did identify all individual The data acquisition system described by Johnston et chromosomes by means of their relative DNA content. al. (14)uses multiplexed analog inputs and a single- Furthermore, additional spatial information is availchannel waveform digitizer. This reduces the data ac- able. Under the assumption that particles exhibiting quisition rate by more than a factor of two compared to low fluorescence intensities but strong light scatter sigparallel processing as described in this paper. Further- nals are preparation artifacts (201, the chromosome data more the multiplexer approach requires precise timing may be separated form the list mode data by creating a if the correlation of the two signals is not to be jeopard- new datafile using the WINDOW software (25). Other ized. parameters such as fluorescence pulse width (chromoOur system digitizes two analog signals in precise some length), location of the centromere, and centrocoincidence. The data are stored in separate bits of the meric index may be calculated as a function of same memory location. The upper byte and the lower fluorescence integral (DNA content) of these data exclubyte of each 16-bit dataword therefore hold synchronized sively. Using the WINDOW program, signals may be information. By means of a n addressing mode using classified according to their relative fluorescence. For byte-pointers, the programmer of Data General’s Eclipse example all #1 chromosomes may be assembled in a computer has easy access to all individual data stored separate data file. The isolated signals may be displayed on the screen before they are further processed. in the main memory.

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At a given digitization rate and flow velocity, small 4. Collard JG, Tulp A, Hollander JH,Bauer FW, Boezeman J Separation of large quantities of chromosomes by velocity sedimentachromosomes will be sampled with a smaller number of data points than larger chromosomes. The coefficient of 5. tion at unit gravity. Exp Cell Res 126: 191-197, 1980. Cosso F, Zweben S J Analog delay line based multichannel digivariation for the small chromosomes will be signifitizer. Rev Sci Instr 56:482-483, 1985. cantly higher and, in some cases, will prevent discrimi- 6. Eisert WG, Nezel M: Internal calibration to absolute values in flowthrough particle size analysis. Rev Sci Instr 49:1617-1621, nation or counting of certain, individual autosomes. For 1978. the recording of karyograms of human cell lines, the 7. High resolution optics combined with high spatial analog processing system will be superior to a 64-dataity in flow. Cytometry 1:254-259, 1981. point sampling scheme. However, the advantage of high 8. Gerlach B, Solleder E, Haucke M, Harms H, Schmid M, Aus HM: resolution with the analog integrators commonly used Application of a high-resolution TV-microscope system to estimate the sequence of centromer separation in Muntjac chromosomes. for recording of flow karyograms is offset by the disadCytometry 5:562-571, 1984. vantage that information on spatial distribution of con9. Gray JW, Lucas J, Pinkel D, Peters D, Ashworth L, Van Dilla MA: stituents is lost. The results shown in Figure 4 Slit-scan flow cytometry: Analysis of Chinese hamster M3-1 chrodemonstrate that our system is as good as the analog mosomes. In: Flow Cytometry IV,Laerum OD, Lindmo T, Thorud solution (3,4) with regard to karyograms, if the chromoE (eds), Universitetsforlaget, Bergen, Norway, 1980, pp. 249-255. somes do not span a large size range. In a previous paper we showed that C.V.s are as low as 0.5% with a test 10. Gray JW,Lucas J, Yu LC, Langlois R Flow cytometric detection of abberant chromosomes. In: Biological Dosimetry, Eisert WG, pulse generator (24). Mendelsohn ML (eds). Springer, Heidelberg, 1984, pp. 25-35. Slit-scan flow cytometry offers the possibility of dis- 11. Gray JW,Peters D. Merrill Sr, Martin R, Van Dilla MA: Slit-scan flow cytometry of mammalian chromosomes. J Histochem Cytocriminating morphological parameters of cells or partichem 27~441-444,1979. cles (9,17,26). This is demonstrated by the example in 12. Halamka J, Gray JW,Gledhill BL, Lake S, Wyrobek A J EstimaFigure 6, mapping regions of fast and slow DNA reassotion of the frequency of malformed sperm by slit-scan flow cytociation along the muntjac chromosome # 1 with high metry. Cytometry 5:333-338, 1984. resolution (21). 13. Jancarik J, Kochanski TP: Multichannel analog transient acquisition system. Rev Sci Instr 48:992-996, 1977. On-line digitization and storage of scan profiles has proved to have at least two advantages: 1)more sophis- 14. Johnston RG, Bartholdi MF, Hiebert RD, Parson JD, Cram L S A slit-scan flow cytometer for recording simultaneous waveforms. ticated calculations may be performed on digitized slitRev Sci Instr 56:691-695,1985. scan profiles (C.I., ratio of parameters, studies on spatial 15. Langlois RG, Yu LC, Gray JW, Carrano AV: Quantitative karyotyping of human chromosomes by dual beam flow cytometry. Proc differences along the particles); and 2) information is not Natl Acad Sci USA 793876-7880,1982. lost because of data reduction. 16. Last TA, Enke C G Analog storage register for fast transient The program library SCAN was constructed to accomrecording. Anal Chem 49:19-23, 1977. modate the needs of our slit-scan analyzing system. Be- 17. Lucas JN, Peters D, Van Dilla MA, Gray JW:Centromeric index sides data acquisition, the digital signal processing part measurement by slit-scan flow cytometry. Cytometry 4:109-116, 1983. of SCAN performs operations on stored profiles, which are usually done by analog circuitry (smoothing, dis- 18. Meyne J, Bartholdi MF, Travis GL, Cram L S Counterstaining Human Chromosomes for Flow Karyology. Cytometry 5:580-583, crimination, and simple filtering techniques, baseline 1984. restoration). 19. Ritz GP, Wallan DJ, Morris MD: Microprocessorcontrolled data acquisition system for pulsed laser spectroscopy. Appl SpectrosIt is noteworthy that this approach keeps original data COPY. 32:493-496, 1978. files unaltered during program execution. This allows 20. Sillar R, Young BD: A new method for the preparation of metareevaluation and comparision of altered and original phase chromosomes for flow analysis. J Histochem Cytochem data at any time. 29:74-78, 1981. Although our system uses a minicomputer system for 21. Stockert JC, Lisanti JA. Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ data storage and computation, the slit-scan analyzer DNA denaturation and reassociation. Chromosoma 37:117-130, described may be realized on low-cost 16/32-bit micro1972. computers with little loss of performance. Programs of 22 Stoehr M, Hutter KJ, Frank M, Goerttler K A reliable preparathe SCAN library may easily be altered to match the tion of mono-dispersed chromosome suspensions for flow cytometry. Histochemistry 74:57-61, 1982 special requirements of the microcomputer.

LITERATURE CITED 1. Arndt-Jovin DJ, Jovin TM: Computer- controlled multiparameter analysis and sorting of cells and particles. J Histochem Cytochem 22:622, 1974. 2. Benaron DA, Gray JW,Gledhill BL, Lake S, Wyrobek AJ, Young IT Quantification of mammalian sperm morphology by slit-scan flow cytometry. Cytometry 3:344-349, 1982. Moore DH, Minkler JL, Mayall BH, Van 3. Carrano AV, Gray JW, Dilla MA, Mendelsohn ML: Purification of the chromosomes of the Indian muntjac by flow sorting. J Histochem Cytochem 24:348354,1976.

23. Van den Engh G, Trask B, Cram S, Bartholdi M: Preparation of chromosome suspensions for flow cytometry. Cytometry 5: 108-117, 1984. 24. Weier H, Eisert W G A pulse generator simulating slit-scan chromosome analysis signals. Cytometry 7:98-100, 1986. 25. Weier H, Eisert W G SCAN-A program library for high resolution slit-scan analysis of chromosomes in flow cytometry. Comput Prog Biomed (in press). 26. Weller LA, Wheeless LL: EMOSS An epiillumination microscope objective slit-scan flow system. Cytometry 3:15-18, 1982. 27. Wurster DH, Benirschke K: Indian Muntjac, Muntiacus Muntjak: A deer with a low chromosome number. Science 168:1364-1366, 1970.