US2850239A - Counting device - Google Patents

Description

Sept. 2, 1958 M. L- POLANYI EIAL COUNTING DEVICE Filed Aug. 16, 1952 4 Sheets-Sheet 1 mvzu'toas voumvn S RD BN s mama's.

NICHK L RKLHARD United States Patent Q CGUNTEN 1G DEVICE Michael L. Polanyi, Stamford, Conn, Richard S. ob ins, Ithaca, N. 1., Earnes E. Johnston, Eipringdale, and lvlorden G. Brown, Darien, Conrn, assignors to American Optical (Iompany, Southbridge, lV-iass, a voluntary association of Massachusetts Application August 16, 1952, Serial No. 304,892

8 Ciairns. (Ql. 235-=92} This invention relates to apparatus for counting and analyzing various kinds of microscopic particles appearing in large numbers and in relatively close relation generally in suspension in solution, such as red corpuscles, particles of yeast, cocci bacteria, mold spores and the like, in an automatic manner and at a relatively high rate of speed. The invention also includes a method for use with such apparatus. While the apparatus of the present invention has broad utility for counting and analyzing purposes, its greatest usefulness at the present time is in the field of blood cell counting and blood analysis.

In the medical profession, for example, it is well known that analysis and evaluation of various characteristics of a single small blood sample may be advantageously employed in many ways. For such reasons it is most desirable to be able to accurately determine the red cell population, and the average red cell size of a sample of blood in a rapid and accurate manner. The red corpuscle count in a sample of human blood may vary over Wide ranges depending upon the condition of the patient from whom it was extracted. Also, the size of individual cells within a selected specimen and the degree of opacity thereof may vary appreciably. When an accurate evaluation of cell population, cell size and hemoglobin concentration can be accurately and rapidly determined, hospital technicians, medical researchers and the like, will be greatly aided. The device of the present invention greatly facilitates this work and provides information of extremely high accuracy.

it must be kept in mind While considering the present invention, that the very small size of the red corpuscles in a blood sample or other microscopic particles being analyzed increases the difficulties which must be overcome when accurately and rapidly providing the above mentioned information. The structure of the present invention, however, is capable of providing the desired information accurately and in a matter of seconds and accordingly has been found in practice to be of great utility.

it is, accordingly, an object of the present invention to provide a combined optical and electrical apparatus capable of receiving a sample of fluid containing microscopic particles and rapidly counting the particle population thereof for unit volume as Well as analyzing same for mean particle size thereof.

It is a further object of the present invention to provide an apparatus of the above character employing an improved electronic differentiating means whereby such evaluation of sample containing microscopic particle may be rapidly, accurately and automatically carried out.

It is a particular object to provide a combined optical and electronic device constructed and arranged to rapidly and accurately count a large number of blood cells in a unit volume blood sample and to accurately compensate according to mean cell size to provide an accurate indication of the true cell count of the unit volume.

It is a particular object to provide a combined optical and electronic device constructed and arranged to rapidly 3 sea and accurately count a large number of blood cells in a unit volume of blood sample and to accurately compensate according to absorptive properties of the cells to provide an accurate indication of the true cell count of the unit volume. The method of use of the apparatus is also being claimed.

It is an additional object to provide a combined optical electronic device of the above character which will rapidly and accurately count a large number of blood cells in a unit volume and to accurately compensate according to mean cell size and absorptive properties thereof to provide an accurate indication of the true cell count of the unit volume.

Other objects and advantages of the invention will become apparent from the detailed description which follows when taken in conjunction with the accompanying drawings in which:

Fig. l is a diagrammatic view of apparatus embodying optical, electrical and mechanical means which may be used in carrying out the present invention;

Fig. 2 is a fragmentary view of a scanning disc which may be embodied in the apparatus of Fig. 1;

Fig. 2A is a graph showing light intensity transmitted by the scanning disc plotted against time;

Fig. 3 is a sectional view taken substantially upon section line 33 of Fig. 1 and showing a specimen carrying slide thereof;

Fig. 4 is an enlarged fragmentary view of a portion of the structure of Fig. 3;

Fig. 5 is a block diagram of an electronic system which may be used with the apparatus of Fig. 1;

Fig. 6 is a schematic wiring diagram of the block diagram of Fig. 5;

Fig. 7 is an enlarged fragmentary view of a portion of structure of Fig. 2;

Fig. 8 is a diagram for use in analytically comparing the image of a mean size particle with the size of the apertures employing in the scanning disc of Fig. 1;

Fig. 9 is an interpolation chart for use in obtaining true count values from the actual count values obtained by the apparatus;

Fig. 10 is a block diagram of a modified form of electronic system which may be used with the apparatus of Fig. 1;

Fig. 111 is a schematic wiring diagram of the block diagram of Fig. 9;

Fig. 12A, 12B and 12C are graphs showing wave shapes and pulses which may be obtained at different locations in the electronics block diagram of Figs. 5 and 6;

Fig. 13 is a nomograph for use in interpretation of the information obtained from the counting units of the device; and,

Fig. 14 is a graph which may be used in place of the nomograph for such purposes.

Optical section As stated previously, the apparatus of the present invention may be used for counting and analyzing microscopic particles of different kinds. However, since one of the greatest needs for such an apparatus at the present time is in the analysis of human blood, the detailed description which follows will be first directed primarily to the apparatus as adapted to such use.

Referring to Fig. l in detail, it will be seen that a microscope optical system 10 comprises an objective 11 optically aligned with a microscope slide 12 for supporting a solution containing light absorbing microscopic particles or the like, to be counted and analyzed. The specimen slide 12 is positioned at the front focal plane of said objective and a cover glass 13 is ordinarily used with the slide. It has been found convenient in carrying out the present invention to employ as the slide 12 one somewhat like a conventional haemacytometer commonly provided for blood count purposes. The slide 12, as will be seen in Fig. 3, comprises a pair of accurately finished slightly elevated specimen supporting plateaus 14in. side-by-side spaced relation, with these plateaus being separated and partially surrounded by a moat 15 for receiving excess fluid. l

V The slide 12 is provided with a pair of accurately finished spaced supporting ribs 16 which extend along opposite sides of said plateaus and in spaced relation thereto to accommodate the moat therebetween. The

' ribsare' acarefully controlled distance above the comthe plateaus and the cover plate will be' the objective upon the optical axis 28 of the optical system is a sub-stage condenser assembly 39 comprising lens components 32 and 34. A light source, preferably of the relatively heavy ribbon filament type, arranged to operate on D. C. current is indicated at 36. Arranged to function with this light'source is a lamp condenser 38 having a field stop 40 adjacent thereto.

Thelight source 36 and lamp condenser 38 are so posi .tionedrelative to a reflector '42 and so aligned with the condenser assembly 30 and objective 1 1 that the filament of the light source 36 will be re-imaged: substantially at the plane of the entrance pupil 44 of the condenser and objective combined. At this entrance pupil 44 may be disposed an aperture plate 46 having'an opaque coating 48 thereon and so arranged as to provide an annular light aperture 50 therein. Light rays passing through the aperfture 50 will provide a cone of oblique light rays for illuminating a specimen upon the slide '12 at the plane 20.

The light illiuninating the specimen upon the slide 12 will be in part diffracted and in part absorbed by the red co rpuscle or erythrocyte population in the solution thereon, with the result that improved contrast between each partially light absorptive particle and the lighter background will be produced. While this type of illumination is preferred for electronic counting purposes, particularly fonsm'all partially transmitting particlesbecause'best' operating results have been obtained therewith, it is possible to omit the aperture plate 46 and obtain usable results with ordinary transmitted or direct illumination. 'However, when more direct light which passes through the particle and through the objective,- as isthe casewhen the aperture'plate 46' is omitted, the

eifective' ratio of the light intensity when 'a patricle is in the microscope field area being used at any instant relative to the total unobstructed light of this field area is lessened. V V 7 Particles in fluid on the slide at the object plane will be imaged by the objective 11 substantially at a conjugate image plane 52. At this image plane 52, however, there is provided a relatively thin circular disc 56 for restricting the area of the imagefield which is transmitting energy at any instant, as will be described more fully hereafter. The disc 56 is rotatably carried by a synchronous motor 58 and this motor is arranged to rotate disc 56 at a predetermined uniform speed. The

motor 58 will normally 'be energized'b'y an alternating current source, such as a conventional 60 cycle, 120 volt apparatus couldbe arranged to operate at any other constant speed within a wide range. The disc 56, as better shown in Fig. 2, is provided with a large predetermined number of light apertures 62 substantially equally peripherally spaced from each other a predetermined distance d. These apertures are also equidistantly spaced a radial amount R, from the center of rotation 63 of the disc 56. Each aperture is circular in shape and of an exact predetermined diameter P which, as will be later more fully explained, closely approximates a predetermined numerical relationship to the normal size of blood cells which is substantially 7.2 microns. merical value will differ for other kinds of particles to be counted.)

When the disc 56 is caused'to rotate, as indicated by the arrow 64, the light apertures 62 will be caused to successively sweep across an image field 71 (indicated by dotted lines in Fig. 2) of the objective located at the conjugate image plane 52. Rays 65 coming fromthe specimen on the slide 12, are imaged at 52 and will pass through an aperture 62 and will be intercepted by a 7 lens 66 which is arranged to receive these rays and direct same substantially upon a fixed area of a photo-sensitive cathode 68 of a photoelectric detector such as aphoto- .multiplier or photocell 70'which is connected to electronic the imagefield 71. The extent of useful travel of the aperture 62 while transmitting light will be controlled by the longitudinal dimension E of an aperture 72a in a field stop 72. The field stop 72 is disposedclosely'ad jacent the disc 56. The length of the scannedimage field E is so chosen relative to the size of the apertures 62 and the space between apertures that distance E will be somewhat greater than the diameter F of apertures 62 plus the distance d between apertures 62. The arrangement will then provide, as shown by, the graph of Fig. 2A, during each single scanning increment of the device,

a time period T during which one aperture transmitslight at a certain intensity L followed by a shorter period If during which two apertures transmit substantially double this amount of light L. Thus,- a rectangular wave form 68A may be plotted as shown by Fig. 2A.;

It will be readily appreciated that as each successive light; aperture 62 is caused, during rotation ofthe scanning disc 56, to sweep acrossjthe aperture 72a in field stop 72, the greater part of the object field 73 on the plateau 14 of the slide 12' (see Fig. 4) will be, in elfect,

V scanned by a small area 74 corresponding to the aperture 62 butwhich area74 is of a reduced sizeso thatits diameter will be in inverse proportion to the magnifica tion of the objective 11, times the diameter F ofthe aperture 62. Shown impressed upon this rectangular wave 68A are small depressions 68B indicative of microscopic particles in or partly in the object field scanned by spot 74 and absorbing various amounts of light. In

other words, ifthe objective 11 has a 44X magnification, the lonigtudinal distance e of the object field being' scanned ,upon the plateau 14 willfbe only one fortyfourth of the distance E inthe image field 71 being scanned and 'the small area, .74 which is scanning'the plateau .14 as the disc 56 rotates will have a diamter 1 which is only one forty-fourth of the diameter F.0f the aperture 62.1 The size of thelapertures '62 shouldbe chosen so as to fairly closely bear a definite relation to the normal size of particles which'are to be counted as will be hereinafter described. 1 It should be herenoted that, 'in order to obtain as uniform object field illumination as possible at slide 12, the bulb 36 willbe positioned inthe optical system so the image of its fiat'front surface formed at the plane 44 will have its longer dimension (This nuassqase extending in the same direction as the longer dimension of aperture 72a.

Since it is also desirable to scan an area of appreciable size, in order to obtain a particle count value which will be indicative of the average condition throughout of the fluid being analyzed, the device of Fig. l is provided with an actuating mechanism 30 of suitable construction for moving the stage 19 and the slide 12 thereon horizontally back and forth across the object field of the objective 11. This travel of the slide 12 is indicated by the double headed arrow 82. The actuating mechanism 80 may comprise a suitable reversible synchronous electric motor 83 having a reduction. gearing 84 of high reduction ratio. In the present preferred embodiment of the invention the actuating mechanism is arranged to perform one complete fore and aft operating cycle each time a specimen is to be analyzed.

The motor 83 and gearing 84 are arranged to cause the plateaus 14 on the slide 12 to move entirely across the object field, after which a sensitive snap action switch 86 adjacent a side of the stage and engaged by the stage 19 will cause the motor 83 to reverse the direction of movement of the slide. The reverse movement or travel of the slide 12 will continue until a second normally closed snap action switch 88 adjacent the opposite side of the stage is engaged thereby for interrupting the supply of current to the motor. A push button 90 for initiating such an operating cycle of the motor 82 may be connected in parallel relation with the switch 88 so that it will only be necessary to manually momentarily actuate the push button 90 in order to start each operating cycle. As soon as switch 88 is free a cycle will be completed automatically. Of course the switch 90 could also serve to start motor 58.

By the use of thin opaque coated areas 92 upon opposite ends of the plateaus 14 and coated areas 93 upon adjacent ends thereof as well as an opaque material in the moat between areas 93 an exact predetermined clear distance, which is represented by the letter G plus letter G (see Fig. 3), may be easily controlled. Areas 92 and 93 may be easily increased or decreased during coating to provide this exact clear distance, and thus the desired total length of the area to be scanned may be accurately established. Since the depth of fluid chamber between the plateaus and cover glass may vary slightly, this variation may be readily compensated for by the control of the total distance (G plus G This method is used to keep the volume of sample being scanned constant. Since the depth of the solution on the slide is established by the height of the cover plate 18 above the plateaus 14-, and since the distances G and G between opaque areas 92 and 3, and the width of the scanned area is controlled by the distance between cen ters of adjacent apertures 62, the volume of the solution being analyzed may always be maintained constant.

During operation of the synchronous motor 58 successive light apertures 62 will be moved lengthwise of the field stop 72 at a constant predetermined rapid speed (in a preferred construction 900 sweeps per second) and by the correct approximately equal spacings d of these apertures circumferentially in the disc 56, one relative to the next, it will be possible to arrange the structure so that two successive light apertures 62 will be completely in the field being scanned simultaneously for a time equal to approximately t (see Fig. 2A). The result of this arrangement will be that background light (through the solution when no particle or particles are in the field) will be transmitted through one aperture 62 to the photo tube 7 for a predetermined interval of time T and thereafter approximately double this amount of light will be transmitted therethrough for a shorter period of time t while two adjacent apertures are simultaneously in the field. Since the motors 53 and 33 always operate in fixed relative relation to each other (regardless of what speeds they are arranged to operate at) and since the total volume being scanned is constant, the same volume of material containing particles will be explored during each complete operating cycle.

There has been described above a method by which a volume or" solution may be synchronously scanned in two directions for determining the total particle count of this volume. It should be clearly understood, however, that other methods may be employed for count determination. The above method, in fact, causes the traces 74 of the individual small aperture 62 in the scanning disc to collectively traverse a certain total distance upon slide 12, which taken in consideration with the depth of cell being scanned, is counting a definite volume of solution each time a specimen is being analyzed by the apparatus. A different method which might be employed which would provide as accurate a final count value might be provided by an accurate control of the number of revolutions of the scanning disc (and thus the total number of individual apertures 62 traversing the image field) and without regard to the total area along the slide which is being traversed by the traces 74 of the apertures at the object field. It would be, in fact, possible to move the slide 12 at random to expose various parts of the specimen in the object field and be able to obtain accurate results. Thus, the total count would depend upon the total length of all of the traces efiected by the aperture 62 times the depth of chamber between slide 12 and cover plate 18. Such measurement of total scanning efiected by the aperture disc 56 could be accomplished by allowing a synchronous motor to operate for a certain predetermined period, or alternatively, it might be possible to rotate the motor at any suitable speed and accurately count by suitable means the number of revolutions of the disc to be used.

The problem of counting opaque microscopic particles of a known size on the slide 12 electronically would not be too difficult a problem (when the particles are sufficiently dispersed thereon) if they were all of the same size and if the number of particles were the only information sought for. In such a case the slide could be, in effect, swept successively by the apertures 62 of the rotating disc while the slide is simultaneously moved across the object field of the microscope by the motor 83. Only electrical signals or pulses above a selected value indicating when one half of a particle or more occludes the light of the scanning area 7 would be counted. However, in attempting to accurately count and analyze materials with particles which vary appreciably n size, which vary appreciably in transmission characterlstics and which vary appreciably in an unpredictable way between not definitely known or commonly accepted limits for sample to sample, such as in human blood analysis work, the problem of obtaining an accurate or true count value as well as additional usable information,,such as mean particle size and light absorption properties, is not a simple one.

It will be shown, however, that the size for the aperture 62 in the scanning disc should be carefully chosen in accordance with the normal size of the kind of particles to be counted so that the apertures 62 will closely approximate the size of the image of such normal particles. By careful consideration of the preselected size for the scanning apertures 62 and careful treatment of the information obtained from the electrical signals derived from the phototube 70 due to background lighting when no cell is present in the field to obscure light and due to different amounts of light flux modulation being produced as the individual particles or blood cells in effect traverse the area 74 being scanned and superimpose pips or minute pulses upon this background signal, accurate blood cell counts may be obtained. The electrical apparatus presently to be described may be arranged to simultaneously or successively count at two different known electrical pulse magnitude discrimination levels for obtaining this accurate information,

It may be assumed of course that a large number of particles of varying size will be dispersed at random' within the sample to be analyzed but will be free'from clumping or undesirable overlapping so as to be in the main individually distinguishable and that the individual particles will appear as disc-like objects of nearly uniform optical density. The solution should be diluted to such an extent that ordinarily only a single particle will be traversed by the scanning aperture 62 or partially traversed thereby at a single instant. Consideration should be given to the'size of the scanning area or spot in the object field relative to the size of particles therein, or alternatively, and more conveniently, the size of the apertures 62 in the disc 56 relative to the size fo the images of the particles atthe image field 71, particularly when it is recalled that the size of particles obscuring the light vary and also that such particle images may be substantially totally or only partially intercepted by the scanning apertures.

In Fig. 7 a small portion of the discj5 containing a single aperture 62 is shown and dotted parallel lines H and I are intended torepresent the width of the path normally traveled by'this aperture during rotation of the disc. The dotted circles P P P and Ptrepresent the images of a plurality of particles oi a single sample at the image plane 52. It Will be readily seen that the images are of diiferent sizes, the image P representing an average size or mean size particle for the sample; For simplifying this discussion this average size particle will be taken as equal to a normal size particlew (The sizeof a normal :particle' is well known.)

The image P represents a particle which is greater than average s'me. Image P also' represents an average size particle while image P on the other hand, represents a particle which is less than an average size particle. The sizes of images P and P have been as sumed .to represent approximately the largest and small.

est size particles present in appreciable numbers in the blood sample to be counted and for which the apparatus is arranged to operate. It may be assumed that substantially all particles in the'sample will fall within these two limits. Obviously, all such particles with their image centers between lines H and I will be so positioned as to momentarily block ofl? sufiicient backgroundlight which ordinarily passes'through the aperture 62 .7, thatthe image P 'of'a normal cell has its center inwardly of line H and this will be counted. .P has its center just outside line H and ordinarily Image should not be counted. 1 But since P is sufiiciently larger than normal to make uptor itsdisplacement from line H, it will be counted. image P is outside line H by an 'amountj'equal to the'displacement; of P? but since image P is of a normal size cell it will not :be counted. Image P is located inwardly of line I, as is image P but since P is somewhat smaller than normal it will not be counted. Notwithstanding this spread or variation in size of cells within a single sample the total count from Fig. 7 will always be two. The larger and sma ll- 7 or cells together'will provide the same number of counts as. an equal number of normal cells will provide when less than or greater than this normal cell size within Well recognized limits. While Fig. -7 has beenused to physicallyshow the problem with which the invention deals, it will be hereinafter more fully shown mathematically and graphically how the amount of variations in cell size within-a sample does not materially effect the. count obtained for such sample. For this reason hereinafter consideration will deal With meancell Size for each sample and proper use of this value provides a correction factor which may be used together with the actuai count obtained in arriving at a closer approximation of the true count of the sample being considered.

In the example just mentioned the average size and normal size were considered to be the same. In most cases, however, the mean cell size will deviate somewhat above or below this normal cell size, and accordingly, a correction factor in every case of this type must be obtained and correctly applied. The reason for such a correction is that the apparatus will be constructedyand' arranged and calibrated to give a correct count when a sample having a mean cell size equal to the normal cell size is being counted. However, when a sample has a mean cell size greater than normal too many cells will be counted and a negative correction must be applied. Co nversely, when a sample has a mean cell size less than normal .a positive correction is needed. The electronic counting means previously mentioned can be arranged to transmit an electrical pulse or signal each time a predetermined amount of light'is blocked oii from the photo'- tube 70 by a particle.

It will now be shown that a correction for mean cell.

size can be obtained in the'following manner. The mean size particle for nearly normal blood may be, according best suited for counting particles of this particular typefor class. Circles P and S are shown at their nearest approach to each other and as being nearly the samesize. Circle S has a radius R The letter 0 is the center of circle? and is the center of circle S. L represents 7 The,

this smallest distance between centers 0 and O particle may completely obscure or only partially obscure the aperture image, and accordingly, it mustbe deter mined when the partially obscuring particle will be counted and when it will not be counted and also what size image or scanning spot S and thus what size aperture 62 should'be used therewith.

locatedin equivalent positions. 'Since in'the example just given the spread or variation in cell size within a 'single sample does not introduce any appreciable. error hereinafter reference will be made to the mean cell size of any sample being considered andit should be appreciated thatin each case the-mean size maybe equal to the normal cell size or'may be'various amounts in Fig. 8 the particle P partially obscures the scanning spot or circle S as indicated by the shaded common areas ABD and DBC. The combined'area ABC!) common to.

both circles P and S in terms of percentage of thescanning spot or scanning area being obscured willbe proportional to the percent modulation from the light beam intensity at the phototube 7t) and consequently directly affect the phototube current .or signal being transmitted to amplifier unit A and cathode follower 953 (see Figs.

land 6). I The particle will be counted if the distance of nearest approach L is such that the inequality particle is. completely opaque and thus does not transmit; 1

9 wherein W is the fractional portion of the incident light on the particle and being absorbed by the particle, and ,6 is the arbitrarily set minimum relative signal strength or bias not passed to a scaler unit of the electronic system (to be later described). For fully opaque particles the factor W will equal unity.

To calculate the maximum percentage of modulation due to the particle or cell P which at a given moment is located at a minimum distance of L measured between the centers and 0 of the circles P and S respectively, BD may indicate the common cord, 04 may indicate the semi-angle in the particle subtended by the cord 13]), a may indicate the corresponding semi-angle in the scanning spot S, and A and A may indicate the areas of particle P and of the aperture S, respectively, determined by the segments BCD and BAD. These two segments, it will be noted will be common to both the particle P and the spot S. For convenience, we may let letter and let gamma (7) equal the ratio of particle radius to spot radius. Thus By substituting in Equation 2 for area ABCD its value in terms of a and a and K and 'y we may obtain by simple trigonometric manipulation the following equation:

Fig. 9 represents graphically the relations stated by the above equations. In Fig. 9 is shown a series of '7 curves for values from 0.50 to 2.00 plotted horizontally against abscissa'K values from 0.8 to 0 and vertically against ordinate percentages of scanning spot S obscured over W from 0 to 100 percent, the symbol 7 represents the ratio of particle diameter or blood cell diameter to spot diameter. K represents the minimum distance between particle or cell center and scanning spot center divided by the sum of the cell radius and half of the spot diameter. Those particles whose distance of nearest approach L in terms of K and 'y is smaller than the value where (see Equation 2) will be counted, while all others touched by the scanning spot will not.

Thus given a value for 'y, the ratio of the particle radius R to the scanning spot radius R only those cells closer to the center of the scanning aperture than the value of K at which the curve therefor intersects the bias level line for which the electronic means is set will pass a signal to the counting unit of the associated electronic equipment. In other words, for example, for a value of 7 equal to .7 and a bias level value set at .425 or 42.5% as indicated by the solid line I, only those particles closer to the center of the scanning spot than K=.3, indicated by solid line M, will be counted. But if the bias value is re-set at the .50 level no such particle will be counted regardless of its position relative to the center of the scanning spot.

Since the particles on the slide 12 are arbitrarily distributed, K statistically assumes all values between zero and one the same number of times during a single count involving a large number of particles. (Of course L assumes values which are larger than one but these values are of no particular interest in the present analysis.) Since only those particles will be counted for which K is .30 or smaller in the previous example in Fig. 9, this means that only 30% of all the particles touched by the scanning spot S (74 in Fig. 1) will be counted. If now the number counted is multiplied by 100 over 30 there will be obtained the correct number of particle centers located between the parallel lines H and I indicating an unknown distance equal to the diameter of the scanning spot plus the particle under investigation. It is obvious therefore that with the additional knowledge of the mean or average particle size being counted, it is possible to determine the exact count. This is so because the total length of the scan is known, the depth of solution in the specimen chamber is known and the width of the scanning spot is also known.

From Fig. 9, which comprises a series of curves plotted from tabulated values previously obtained, it is possible to plot a curve which Will give K over Kg values as a function of 'y for a given ratio of C over C wherein K is proportional to the count which would be obtained from a sample at the C bias level, while K is proportional to the count which would be obtained from the same sample under identical conditions but at a C bias level. It follows that in order to obtain the mean or average size of blood particle under examination, it is possible to take two counts at two difierent known bias levels, (for example, .30 and .45) and under otherwise identical conditions and then take the ratio of these two readings and refer to a graph substantially like that in Fig. 14, in which the ratio of is identical with the ratio of the two readings made with the two different biases.

From the knowledge of the ratio (and for the C and C values used) it is possible to determine gamma (7) for the particle being counted. Since '7 is equal to the average cell diameter being counted divided by the scanning spot diameter which is known, the average cell diameter is obtained. Once the 7 value for the particular count is known reference must be made to Fig. 9 and the value of K read from same. This value of K in combination with the right value of c (the proper c setting which takes into account a suitable correction for the transmission characteristic of the particle will be explained later) will give the value of K which is the correction to be used for the particular count.

Electronic section In a preferred embodiment of an electronic system which may be used, the photo-multiplier 70 (Fig. 1) will be carried by a housing 94 containing an amplifier unit 95A and a cathode follower unit 95B coupled through lead 70A to the multiplier (see Figs. 5 and 6). The light intensity when a single aperture 62 is transmitting is arranged to produce an electrical current through a load resistor 70B proportional to the light incident upon the photo-cathode 68 and thus a potential across this load resistor for the photo-multiplier. The current wave form is grahically indicated at 96 in Fig. 12A and relative to a zero dotted line 96'. Its magnitude is equal to 1,, when one aperture 62 is transmitting and 2 I when two apertures are in the field as indicated by substantially uniformly spaced pulses 96", and these amounts are lessened when particles are momentarily in or partly in the field and creating signal pulses shown by pips 96B and 96C. The amplitude and width of each of these pips will depend upon the amount of light blocked off by each of the particles. The instantaneous decrease in light creating each pip, it should be noted, is due to the size of particle being totally scanned or part of particle being scanned as well as the light absorbing characteristics of this particle.

The housing 94 is of relatively small size and, accord ingly, may be attached directly to the supporting housing or equivalent means (not shown) for positioning the optical and mec I 11 com onents of the apparatus. The amplifier unit 95A and the cathode follower 95B offer very low output impedance so that the signal produced by the photo-cathode maybe transmitted without material wave form distortion by means of a flexible scanning during theshorter period'z.

V is passed to an amplifier 111 and amplified to produce an shielded cable 94A to the other electromc parts of lhe equipment and counting or scaler'units' (presently tobe 7 described) which may be .disposed,.forconvenience and.

such, at a distance from the microscope.

The signal shownat 96 inFig. 12A and somewhat amplified is fed simultaneously through two channels whichfnay be conveniently termed 'a pip channel '97 and a compensation control channel 93;: In the channel 98 the voltage wave form is passed by lead 9% to a high Q resonant frequency selector of filter network 99 which in the present preferred construction is tuned to 900 cycles 'per second. Thisnetwork is arranged to give a pure sine wave with all harmonics thereof removed and the. pips 96B and 96C and pulses 96 removed as shown at 99 in Fig. 12C. 'The sine wave is then further am- 7 plified by stage 101 and the output of this amplifier stage 7 is fed to a bias: control unit 103 which has a manual control 103 adjustable before each run to give an output amplitude equalto or a little greater than the maximum signal or pip to be encountered in. the pip channel 97; Unit'103 also has a readily adjustable control 13A which'may be moved from its-#1 position to its #2 or #3 positions for different bias levels. ciated that if position #1 gives a 100% voltage drop across resistors 103R 103113 and 103R to ground, positions #2 and #3 will give fractional parts thereof proportional to the resistances 103R and 103R in the one case and proportional'to resistance 103R in the other.

7 The output from'the bias control stage 103' is amplified and passed through a. full wave rectifier. unit 105 and the average Di, C voltage derived therefrom and used as a negative bias, is passed through a conductor 107 onto the plate of an'amplitude discriminator diode 107 in the compensation control channel 97. The reason for this negative bias is to maintain the discrimination level of the diode 107 constant notwithstanding small longrterm changes in light level or output from the light source 36 or response characteristics of photo-multiplier or power supply voltage'for the multiplier should such occur.

The signal 96 in the pip channel 97 coming from the grid of. the cathode follower 95B isfed to a diode clipper. 109, which clips off the negative portion of the wave form 96 below dotted line E and results in a'wave form 109A. Clipped portions 109B are indicated by dotted lines. 'This clipped off portion at 1093 is due to the double aperture The signal. 109A inverted wave form 111A. This wave form is clamped by diode-clamper 111' at a D. C. level 111B. 7 This clamped wave shape 111A is fed to. the amplitude discriminator 107 for pulse height selection. Thisdiode 107 passes only positive pulses representing blood cells larger.

than a predetermined minimum amplitude shown by dotted line 107A thereby producing a wave form 107B containing fewer pulses than contained in the partly dotted and partly full line wave form 107C which it received.

The wave shape frorn the discriminator stage 107 is shown at 10713 and fur'theraniplified by an amplifier 113 as shown by wave form 113A; This wave form 113A is clamped by a diode clamper 115 at the base line 113B.

The pips 113C of'this wave form are usedto trigger a one-shot multivibrator 117 whoseroutput signal containing pulses of equal amplitude and width is shown at 1 17AQ This signal is fed in turn directly to an electronic decade 'scaler unit of known construction 119. From this 7 sealer unit 119 a numerical count will be obtainable and this count maybe at times used directly as an approximate indication 'of red blood cell count conditions or used with avariable corrective factor to obtain a trueqred blood cell count as will be later described.

By the use of control 103A, a plurality 'ofbias levels may be used toobtain counts of different magnitudes'for It will be appre counts N and N on the decadescaler 1 19 and it might be. convenient to arrange the support .19 [of Fig. 1 .to actuate the control 103A so as. to count at one bias level a while the support is moving in one direction and so as to count at the other bias level during its reverse travel.

The decade sealer could be equippedf-iffdesired, with means arranged to record both counts. Or alternatively i a pair of decade sealers could be alternately used for the successive counts.

At times it may be desirable to additionally provide a rate meter unit for indicating approximate red blood cell population and this may be accomplished by. also taking signal 117A and feeding it through a cathode'fol-.

' lower 121 to a rate meter 123. The ratelme ter is essen-.

tially a voltmeter but differently calibrated and arranged to read the average voltage being applied theretoby the cathode follower 121 and signal-117A applied thereto. The pulses from the multivibrator 117 are all of a constant amplitude and pulse width and thus the reading on the rate meter 123 is' dependent only upon the number of pulses per given unit of time. Of course, when desired a sealer-unit 119 might be omitted and only readings from the meter 123 used but a lower degree of accuracy would result.

A modified form of electronic system is shown by Figs. 10 and 11 and whilethe photo-multiplier 70, amplifier unit A and cathode follower 95B are the same as used in the system of Figs. 5 and ,6, the modified system ineludes additional integrating electronic components for. obtaining other information from the specimen containing microscopic particles'being counted and analyzed, The signal pulse 96'from the grid of the cathode follower 95B may be divided (see Fig. 11) and used in the two pip channels 97A and 97B. In the pip channel 97A, ai

diode clipper unit 139, amplifier unit 141; diode clamper 141', amplitude discrimination 142, amplifier 142, diode clamper 145 and multivibrator 147 are like similar units in pip channel 97 of Fig. 6.

'As previously, av signal may be taken from cathode of the cathode follower unit, through a lead 98 for use i inra compensating control channel 93 The compensation control channel 98 however, passes a signal to the filter network 99 and then to amplifier unit 101 and to a bias control stage 133' which has a readily controllable f member 133A which may beset at its #1 position while the control 133 is adjusted to its .level (similar to control 103. in Fig. 6). Thereafter control 133A i be set at its position to give a preselectedpercent level such as an 80% bias level. -This signal is amplifiedand 5 rectified by unit 135 to provide a D. C. bias 'E between point 135 and ground. 3

The rectifier unit 135 is provided with an adjustable control 136 so that a'diiferent D. C. voltage derived from a voltage divider 135A may be passed by conductors 137A and 13713, respectively, to pip channels 97A and 97B. Substantially, similar 'units (139B, 141B, 142B, 141B, 143B, 145B and 147B) are contained in channel 9713 as those already mentioned in channel 97A.- The two D. C. voltages from divider 135A are for controlling the amplitude discrimination'levels of the two amplitude discriminator diodes 142 and 14213. The remainder of 7 pulse signal N islalsotaken off at 1483 and .used in an a single specimen during successiveruns. In'fact, a pair 'of counts at different known bias levels, say 80%' and 35%, as set by the control 103A may be used to obtain anti-coincidence circuit 149 of known type." The signal pulses in the two pip channels 97A and 97B are fed into the anti-coincidence circuit'which will only give: an out put pulse when either signal appears but not when both appear simultaneously. They cancel each other. The output from this unit 149 is supplied to a second decade counter 150 which will then give a count N which is in effect the difference between N count and N count ob tained simultaneously.

From the N and N N wines from either electronic system there is obtained the ratio of needed for average size determination.

Selection scanning aperture size The optimum scanning aperture size may be determined from inspection of Fig. 9, keeping in mind that the operating region selected must be such that the distance as a function of K between the 7 lines should be as constant as possible. This is important because by so doing the spread of size distribution effect on count is best minimized. The other requirement is that the changes of v in functions of the ratio of Use of device When the device is to be placed in service in the counting and analyzing of blood the following technique may be used: A sample of blood may be obtained from a puncturing of a finger or ear lobe in the normal clinical fashion and diluted in a standard pipette with a conventional fluid (such as Hayems solution) to the correct proportions, preferably 1 part blood to 200 parts of solution. A portion of the prepared sample is applied to the slide 12 and the cover plate 18 placed thereover. This assembly is then placed upon the movable support or stage 19 with the coated areas 92 and 93 spaced from each other in the direction of movement of the stages as indicated at 82.

Since two different counts at different known percentage bias levels are to be made and are to be based upon the total number of pulses exceeding these known percentages of the maximum signal height obtainable, it is essential to adjust the control 103' while the disc 56 is rotating to the position where the decade sealer just ceases to count pulses with control 103A set in its #1 position. This establishes a 100% reference or bias level. The control 183 may be set at a bias level so that pulses pass to the scaler units and then gradually adjusted until no pulse passes to the scaler. Once the maximum pulse height or 100% level is established, it is a simple matter to set the bias control 103A to its #2 position, start the stage motor 83 and make a run and then set the control at its #3 position and make a second run. These runs would be at known percentages of the total resistance 103R 103K and 103R being used. As previously stated, two levels may be successively set manually or automatically when the single pulse channel system of Fig. 6 is being used to obtain counts N and N The slide 12 will be moved to its extreme right hand position when a count or run is to be made. And, of course, at this time the decade scaler 119 will be set a zero. The bias control 103A is switched to its #2 position (for example its 45% bias position), a run is made i and a reading taken on the sealer 119. Switch 103A is then shifted to its #3 position (for example its 30% bias setting) and a run of the slide 12 across the object field of the instrument in the opposite direction made. A second count is taken. The ratio of one reading to the other reading is now to be used. For this discussion, a ratio of the larger reading N (at the lower bias level) to the smaller readingN (at the higher bias level) will be used.

On the diagram of Fig. 14 (which has the 2 ratio values a t LE) bemg equal to 2 for the two readings on the horizontal scale and gamma (7) values (equal to ratio on the other scale in function of bias level C and bias level C take a '7 value as indicated by line 157 corresponding to the average or mean size cell counted, and with this value and the value of the bias used obtain from the diagram of Fig. 9 a value for K for the particular count under consideration.

From the basic calibration curve for the instrument (the instrument being calibrated for normal size cells) read the normal count corresponding to normal size particles. Multiply the normal count by correction factor obtained from Fig. 9 and by an additional correction factor spot, radius+ normal radius spot radius-l-avrage radius to obtain the correct number of cells per unit volume.

correction is a detection error while the additional factor is a volume error correction). These two factors together should correct the actual count so as to give a true count for the solution being counted. A nomograph such as shown in Fig. 13 having N and N values and mean diameter values may be used for any preselected pair of bias levels and preselected scanning aperture in place of the above mentioned procedure and curves. In order to obtain the average size from the readings N and N A straight line 154 extending through obtained values for N and N will give a mean diameter value on scale 155. If either the scanning aperture size or the bias levels were changed, obviously a different nomograph would be required.

When using the two pip channel systems of Figs, 10 and 11 with anti-coincidence circuit 149, values N and N may be obtained and used; N being in effect equal to the N count minus the N count. These Values may be used in a similar manner, or a nomograph (not shown) using N and N drawn to provide mean diameter values.

While particles in solutions or fluids have been discussed in the preceding disclosure it should be understood that dust particles and the like not in solution also may be counted and analyzed in like manner. While red cells have been discussed primarily in connection with this equipment, white cells may also be counted. For this count the red cells would be removed by hemolysis and the nuclei of the white cells, which fortunately are close to the same size as the red cells, would be stained or alternatively the illumination system changed to give high contrast between nuclei and the rest of the cell. For some specimens a true phase microscope optical system would be used in place of the system shown in Fig. 1.

Suitable mechanical recording'means could be provided a to respond to the number, of pulses (N and N being obtained during successive runs or during simultaneous runs and to automatically indicate (in place of the memograph of Fig. 13) the true count value for the fluid being counted or the mean diameter of the particles thereof.

Having described our invention, we claim:

1. An optical system for use with electronic counting.

means for rapidly and accurately determining the'num- 'her of microscopic particles dispersed Within a predetermined unit volume of a specimen of a preselected type,

said optical system comprising an objective having an object field and an image field conjugate thereto, a field stop in fixed position adjacent said imagetfield and optically aligned with said objective, a support for positioning a specimen in said object field, a light source and" rapid succession and at a predetermined uniform speed. through said image field and into and out of optical alignment with said field to stop and objective, said scanning means being arranged to operate concurrently with the.

transverse movement of said supporttand in a direction angularly disposed relative to the direction of movement of said support, the spacingbetween centers of adjacent scanning apertures in said scanning means being no, greater than the dimension of the light aperture in'said field stop taken in the direction of movement of said scanning apertures and no less than one half a specimen on said support whereby light fromsaid specimen at said object field will be passed through at least one and no more than two scanning apertures at any time during use of the optical system for reception by saidelectronic countingmeans.

, 2; An optical system means for rapidly and accurately determining the num ber of microscopic particlestdispersed within a prede-j termined unit volume of a specimen of a preselected type, said'optical system comprising an objective having an object field and an image field conjugate thereto, a field stop in fixed position adjacent said image field and optically aligned with said objective, a support forpositioning a specimen'in said object field, means forming a light source of substantially annular contour and condenser I aligned therewith for conically illuminatingthe specimen when positioned on said support in said object field, means for movingsaid support transversely with espect to the optical axis of said optical system, opaque nning means having a series of substantially equally ed'scannin apertures of predetermined size therein, redetermined size of each scanning aperturebeing i relatcdin size to the sizeof the image of an e size particle of said preselected type of specineans for'actuating said scanning means for movaid scanning apertures in rapid succession and at a predetermined uniform speed through said image field i'intoand out of optical alignment with said field stop objective, said scanning means being arranged to ate concurrently with theftransverse movement of suppcrt and in atdirection angularly disposed relatothe direction of movement of said support, the

tive spacing between centers of adjacent scanning apertures insaidscanning means being no greater thanthe di mension of the light aperture in said field stop taken in V r the direction of movement of said scanning apertures and no less than one half said dimension, whereby light from ssures for use with electronic'countingf a specimen on said support at said object field will be passed through at least one and no more than two scanning apertures atall times during use of the optical system for reception by said electronic counting means.

3; An optical system for use with electronic counting means for rapidly and accurately determining the number of microscopic particles dispersed within a predetermined unit volume of a'specimen of a preselected type, said optical system comprising an objective having an 7 object field and an image field conjugate thereto, a field stop in fixed position adjacent said image field and optically aligned with said objective, a support for positioning a specimen in said object field, a light source and condenser means for illuminating the specimen when positinned onsaid support in said object field, means for moving said support transversely with respect to the optical axis of said optical system, opaque scanning'means having a series of substantially equally spaced scanning apertures of predetermined size therein, the ratio of the diameter of the image of an average size particle of said preselected type of specimens to the diameter of each scanning aperture being within a range of ratios between approximately 1.25 and approximately 0.8, means for j actuating said scanning means for moving said scanning apertures in rapid succession and at a predetermined uniform speed through said image field and into and out of optical alignment with'said field stop and objective, said scanning means being arranged to operate in predetermined timed relation to the transverse movement of said support and in a direction angularly disposed rela tive to the direction of movement of said support, the

spacing between centers of adjacent scanning apertures in said scanning means being no greater than the dimension of the light aperture in said field stop taken in the{ direction of movement of said scanning apertures andv no less than one half saiddimension, whereby light from a specimen on said support at said object field will be passed through at least one and no more than two scanning apertures at all times during use of the opticalisysv. tem 'for reception by said electronic counting meansi e '4. A device for use in rapidly'and accuratelytdeter-t mining the number of microscopic particles dispersed within a predetermined unitvolume of a specimen of a preselected type, said; device comprising an optical system including an objective having an object field and an image field conjugate thereto, a support for'positioning a specimen in said object field, light source means 'for illuminating said specimen when positioned on said'support in said object field, means for moving said support transversely with respect to the optical axis of saidoptlcal system, scanning means having a series of scanning V apertures of predetermined size arrangedto be moved in'rapid succession through said image filed, saidrscair.

ning means being arranged to operate concurrently with' the movement of said'support and in a direction angular- 1y disposed relative to the direction of movement of said support, said predeterminedsize of each scanning aperture being closely related in size to the size of the image of an average size particle" of said preselected type of specimen, wherebylight flux modulation of appreciable value will be produced in the light passing beyond'said scanning means when microscopic particles in said speci men on said support are being scanned, photoelectric means optically aligned with said objective so as-to receive the modulated light and provide an electrical signal theinstantaneousmagnitude of which is substantially proportional to the amount of light beingreceived thereby, an electronic network for amplifying said signal,

first electrical means in said electronic network for passing only those electrical pulses due to particles being scanned which have electrical amplitudes greater than a first preselected value, second electrical means in said network for passing only those electrical pulses due to said particles being scanned which have electrical ampli tudes greater than a second difierent preselected value,

17 and first and second counting means connected to said first and second electrical means, respectively, for separately counting the total number of pulses passed by said first electrical means and the total number of pulses passed by said second electrical means during a single complete cycle of operation of said device.

5. A device for use in rapidly and accurately determining the number of microscopic particles dispersed within a predetermined unit volume of a specimen of a preselected type, said device comprising an optical system including an objective having an object field and a image field conjugate thereto, a support for positioning a specimen in said object field, light source means for illuminating said specimen when positioned on said support in said object field, means for moving said support transversely with respect to the optical axis of said optical system, scanning means having a series of scanning apertures of predetermined size arranged to be moved in rapid succession through said image field, said scanning means being arranged to operate concurrently with the movement of said support and in a direction angularly disposed relative to the direction of movement of said support, said predetermined size of each scanning aperture being closely related in size to the size of the image of an average size particle of said preselected type of specimen, whereby light flux modulation of appreciable value will be produced in the light passing beyond said scanning means when microscopic particles in said specimen on said support are being scanned, photoelectric means optically aligned with said objective so as to receive the modulated light and provide an electrical signal the instantaneous magnitude of which is substantially proportional to the amount of light being received thereby, an electronic network for amplifying said signal, adjustable bias control means in said electronic network for establishing a maximum bias level which is substantially equal to the maximum pulse amplitude due to particles being scanned, first and second fractional bias selecting means in said network for selecting first and second biases equal to two difierent known fractional portions respectively of said maximum bias, and for passing respectively only those electrical pulses of amplitudes greater than said selected biases, and first and second counting means connected to said first and second fractional bias selecting means for separately counting the number of pulses, representative of the particles, passed by said last-mentioned means at said first fractional bias level and at said second fractional bias level respectively.

6. A device for use in rapidly and accurately determining the number of microscopic particles dispersed within a predetermined unit volume of a specimen of a preselected type, said device comprising an optical system including an objective having an object field and an image field conjugate thereto, a support for positioning a specimen in said object field, light source means for illuminating said specimen when positioned on said support in said object field, means for moving said support transversely with respect to the optical axis of said optical system, scanning means having a series of scanning apertures of predetermined size arranged to be moved in rapid succession through said image field, said scanning means being arranged to operate concurrently with the movement of said support and in a direction angularly disposed relative to the direction of movement of said support, said predetermined size of each scan ning aperture being closely related in size to the size of the image of an average size particle of said preselected type of specimen, whereby light flux modulation of appreciable value will be produced in the light passing beyond said scanning means when microscopic particles in said specimen on said support are being scanned, photoelectric means optically aligned with said objective so as to receive the modulated light and provide an electrical signal the instantaneous magnitude of which is 18 substantially proportional to the amount of light being received thereby, an electronic network for amplifying said signal, first electrical means in said electronic network for passing only those electrical pulses due to particles being scanned which have electrical amplitudes greater than a first preselected value, second electrical means in said network for passing only those electrical pulses due to said particles being scanned which have electrical amplitudes greater than a second different preselected value, first and second counting means connected to said first and second electrical means, respectively, for separately counting the total number of pulses passed by said first electrical means and the total number of pulses passed by said second electrical means during a single complete cycle of operation of said device, and additional counting means connected to said first and second counting means for counting only the number of pulses which are counted by one of said counting means and not counted by the other of said counting means.

7. A device for use in rapidly and accurately determining the number of microscopic particles dispersed within a predetermined unit volume of a specimen of a preselected type, said device comprising an optical system including an objective having an object field and an image field conjugate thereto, a support for positioning a specimen in said object field, light source means for illuminating said specimen when positioned on said support in said object field, means for moving said support transversely with respect to the optical axis of said optical system, scanning means having a series of scanning apertures of predetermined size arranged to be moved in rapid succession through said image field, said scanning means being arranged to operate concurrently with the movement of said support and in a direction angularly disposed relative to the direction of movement of said support, said predetermined size of each scanning aperture being closely related in size to the size of the image of an average size particle of said preselected type of specimen, whereby light flux modulation of appreciable value will be produced in the light passing beyond said scanning means when microscopic particles in said specimen on said support are being scanned, photoelectric means optically aligned with said objective so as to receive the modulated light and provide an electrical signal the instantaneous magnitude of which is substantially proportional to the amount of light being received thereby, the spacing between centers of adjacent scanning apertures being such as to present to the light transmitted by said objective at all times during operation of the device an effective scanning area which is equal to the area of at least one scanning aperture, an electronic network for amplifying said signal, adjustable bias control means in said electronic network for establishing a maximum bias level which is substantially equal to the maximum pulse amplitude due to particles being scanned, first and second fractional bias selecting means in said network for selecting first and second biases equal to two difierent known fractional portions respectively of said maximum bias, and for passing respectively only those electrical pulses of amplitude greater than said selected biases, and first and second counting means connected to said first and second fractional bias selecting means for separately counting the number of pulses, representative of the particles passed by said last-mentioned means at said first fractional bias level and at said second fractional bias level respectively.

8. A device for use in rapidly and accurately determining the number of microscopic particles dispersed within a predetermined unit volume of a specimen of a preselected type, said device comprising an optical system including an objective having an object field and an image field conjugate thereto, a support for positioning a specimen in said object field, light source means for illuminating said specimen when positioned on said support in said object field, means for moving said support transversely with respect to the optical axis of said optical system," scanning means having a' series of scanning apertures of predeterrnined size arrangedto b'e moved in rapid succession through said image field, said scanning means being arranged to operate concurrently with the movement of said support andin a direction angularly disposed relative to the direction of movement of said support, said predetermined size of each scanning aperture being closely related in size to the size of; the image of an average size particle of said preselected type of specimen, whereby light flux modulation of appreciable value will be produced in the light passing beyond said scanning means when microscopic particles in said specimen on said support are being scanned, photoelectric ,means optically aligned with said objective so as to receivethe modulated light and provide an electrical signal the instantaneous magnitude of which is sub- 'stantially proportional to the amount, of light being received thereby, the spacing between centers of adjacent scanning ap erturesbein'g such as to present to the light transmitted by said objective at'all times during operation1of thedevice an eifective scanning aperture which is alternately equal to the area of one scanning aperture j and an area appreciably greater than the area of one scanning aperture, an electronic network for amplifying said signal, adjustable bias control means in said'electronic network for establishing a maximum bias level which is'substantially equal to the maximum pulse amplitude due to particles being scanned, first and second fractional'bias selecting means in said network for selecting first and second biases equal to two difierent known-fractional portions respectively of said maximum bias, and for passing respectively only those electrical pulses, of

7' amplitudes greater than said selected biases, and-first and second counting means connected tosaid first and second fractional bias selecting means for separately count- 7 ingthe number of pulses, representative of the particles passed bysaid last-mentioned means at said first fractional bias'level and at said second fractional.biaslevel-respectively.

7 References Cited the file of this'patent V UNITED STATES PATENTS 1 2,393,631

OTHER REFERENCES The Model 200 Pulse Counter by Higinb'otham et al.,

from .The Review of Scientific Instruments," volume 18, No, 10, October 1947. I j

Rev. Sci. Inst, volume 22,, N0. 3, single Channel Analyzer by Francis et al. (pages 133-137), March 1951. a i r

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