US2847162A - Counting of particles - Google Patents

Description

Aug; 12, 1958 E. w. MEY 2,847,162 r poummc 0F PARTICLES.

. Filed 001:. 31, 1952 I 4 sheets-sh t 1 Aug. 12, 1958 E. w. MEYER COUNTING OF PARTICLES 4 Sheets-Sheet 2 Filed Oct. 31, 1952 QM N mm Aug. 12, 1958 l E. w. MEYER COUNTING 0F PARTICLES 4 Sheets-Sheet 3 Filed Oct. 31, 1952 gv S m mw N l H \\M\\\ \W\\ \w n m \m m% "5 am 1 g \UNM n A U .w -m WH H P... W E Q Mm 1 g Q, Q Q mm 3 is as Aug. 12, 1958 4 Shets-She et 4 Filed Oct. 31, 1952 United States Patent CQUNTING 0F PARTICLES Ernest William Meyer, St. Margarets, England Application @ctoher 31, 1952, Serial No. 318,110

Claims priority, application Great Britain November 2, 1951 6 Claims. Cl. 235-92 This invention concerns improvements relating to the counting of small particles, such for example as blood cells. Particle-counting apparatus is known in which, a distributed sample of the particles. is scanned over a known area and the particles are counted automatically, provision being made for compensating for the error arising from the inclusion in the count of part imagesappearing at the edges of the said area. It is an object of the invention to provide simple but effective apparatus of this kind free from practical difficulties associated with known apparatus.

According to the invention, apparatus for counting such particles comprises a microscope arrangement for scanning a sample of the particles, a mask through which the sample is scanned over a known area and which has an aperture only slightly wider than the magnified image of the largest particle that it is desired to detect, a mask through which the sample is simultaneously scanned and which has an aperture whose width is only a small frac-.v tion of the width of the first-named aperture, particledetecting means disposed beyond each of the apertures in the said masks and responsive to the presence of particle images magnified by the said microscope, and counting means operatively connected to the said particledetecting means for counting the said images. Particu, larly, if the distribution of the particles in the sample is not of a completely random nature, the narrow zone scanned through the second aperture should be immediately adjacent to the main zone scanned through the first aperture. The second or guard aperture is intended to detect only the edge effect and may, for instance, have a width of about one twentieth of the main aperture. However, if the particle distribution is perfectly random, an effect equivalent to the edge effect can be obtained by scanning a like narrow zone not immediately adjacent tothe mainzone.

With such apparatus, a substantially true count of particles in a known area represented by the main zone scanned can be estimated from the difference between the numbers of images to which the detecting means beyond the main and guard apertures have responded. This difference may be directly registered by counting means of well known kind.

A preferred manner of carrying the invention into effect will now be described with reference to the accom-- panying drawings, in which:

Figure 1 is a diagram illustrating the apparatus, Figure 2 a plan view of a microscope stage used in the apparatus, Figure 3 a longitudinal section through the said stage, Figure 4 a cross-section on the line IV-IV in Figure 3, and Figure 5 a detail view in end elevation illus-- trating a part of the mechanism. Figure 6 is an electrical circuit diagram for certain components of the apparatus shown in Figure 1, pulses hereinafter referred to being conventionally indicated at various stages.

Referring to Figure 1, light from a source 1 of stabi-- lized intensity is directed by an optical system 2, including a condenser 3, through a mechanically operated scanning stage 4 adapted for supporting a slide 5 carrying a sample in which particles are to be counted. Beyond the stage 4, the light passes through a microscope 6, comprising objective 7 and eyepiece 8, and falls upon a semi-silvered mirror 9, or a Swan cube 9', arranged at an angle of 45 to the incident light. This is adapted, in well-known manner, for transmitting a part of the light and for reflecting a part of the light at right angles. The transmitted light passes through an aperture 10 in a mask 11 and falls upon a photo-electric cell 12 of the known photo-multiplier type. The reflected light passes through an aperture 13 in a mask 14 and falls upon a similar photo-multiplier 15. The main aperture 10 is square and only slightly larger than the image of the largest size particles it is desired to detect. The guard aperture 13 has a width which is only a very small fraction, for example one twentieth, of the width of the main aperture 10 and the narrow image zone received by it is immediately adjacent one marginal edge of the image zone received by the said aperture 10. In the direction of scan, however, the aperture 13 has a length slightly larger than that of the main aperture 10, the arrangement being such that images common to both apertures tend to be first detected at the aperture 13.

With this arrangement, the photo-multiplier 12 will respond to images at the aperture 10 of particles appearing in the eyepiece 8 of the microscope 6 and will give a voltage output in the form of voltage pulse's substantially proportional to the sizes of respective images of the whole or parts of particles. The photo-multiplier 15 will produce an output of voltage pulses corresponding to images at the aperture 13. These outputs are fed through respective D. C. amplifiers 16, 17 and thermionic amplitude-discriminatory or trigger circuits 18, 19 to counting means comprising a gate circuit 20 and a regiSter or meter 21, these components being of per se wellknown type. In this embodiment, the circuit 18 is designed to transmit all pulses received, but the circuit 19 to transmit only pulses corresponding to complete obscuration of the guard aperture 13. Thus the circuit 18 I transmits a number (N of pulses corresponding to the number of particles and parts of particles detected in the whole known area consisting of the main zone scanned through the aperture 10. The circuit 19 transmits a number (N of pulses corresponding to the number of particles materially overlapping the marginal zone scanned through the aperture 13. The register 21 records the difierence (N N between these numbers. From this diflerence, with a knowledge of the length (L) of scan and the widths (W W of the apertures 10, 13, it can be shown that a count it of the number of particles in the known area scanned can be obtained by the equation Figure 6 illustrates by way of example known forms of circuit suitable for the components 18-21. In this example, circuits 18 and 19 are so-called Schmitts amplitude-discriminatory circuits. These circuits can be adjusted by means of potentiometers R and R with respect to the sizes of the pulses, received from the amplifiers 16 and 17 respectively, by which they will "be activated. The gate circuit 20 is of the kind comprising a suppressor gated pentode Vg. The output of pulses from the circuit 18 is applied to the control grid and the output of pulses from the circuit 19 to the suppressor grid of the valve Vg. From the explanation given above, it follows that a pulse may be received by the gate circuit 20 from the circuit 18 only, whereas, a pulse from the circuit 19 will always be accompanied by a pulse from the circuit 18. A negative pulse from the circuit 19 will cut off the valve Vg, so that the positive pulse also received from the circuit 18 will be blocked. Only a pulse from the circuit 18 without a coincident pulse from the circuit 19 will be passed by the gate circuit 20 to the register 21. Consequently only the excess (N -J) of pulses received from the circuit 18 over the number of pulses received from the circuit 19 is passed to said register.

For scanning a sample, it is necessary to move the slide 5 in relation to the microscope 6 in a systematic manner, commonly in linear or spiral fashion. Linear scanning is preferred, but it is an advantage of apparatus in accordance with the invention that the amplitude of the pulses produced is not affected by variation in the speed of scanning or even by the stage 4 being at rest. Consequently, the discriminatory circuits can be preliminarily adjusted with the stage at rest. Figures 2-5 illustrate a mechanical stage whose use has advantages for the present purpose. As shown in Figure 3, the slide 5 rests upon the upper stage platform 22 across an opening 23 therein, the platform 22 resting in turn upon a lower platform 24 with an opening 25. The platform 24 is movable longitudinally of a fixed base 26, whilst the platform 22 not only partakes in that movement but is also movable transversely, the transverse movement providing the linear scan and the longitudinal movement the interline shift at the end of each line. The platform 22 is supported at three points by balls 27 running in transverse V-grooves 28 formed inthe two platforms. On one ball 27, however, the platform 22 is supported by a plane surface 29, so that relative expansion effects cannot cause constraint. Adjustable stops 30 serve to limit the transverse movement of the platform 22. The platform 24 is also supported at three points by balls 31 running in longitudinal grooves 32 formed in the said platform and the base 26, but again support at one point is by a plane surface 32 (Figure 4). The platform 24 is constantly drawn to the left in Figures 2 and 3 by a tension spring 33, so that a stop plate 24' thereon is held in contact with a ball abutment 34 carried by a screw 35 which is mounted in a bracket 36 on the base 26 and is operated as hereinafter described for producing the longitudinal movement of the platforms.

Again to avoid possible constraint and also possible disturbance of the accurately focussed position of the slide 5, the platform 22 is not mechanically connected to the means for imparting transverse movement to it. Instead it is actuated magnetically across an air gap 37 by the effect upon a longitudinally elongated armature 38 attached to its underside of a horseshoe permanent magnet 39 which is carried in an upstanding bracket 40 supported from the base 26 by long parallel links 41. These links 41 are pivotally mounted at 42 and pivotally connected at 43 to a cross bar 44 on which the bracket 40 is fixed. For its additional support, the bracket 40 has fixed to it an angle piece 45 resting by a ball 46 on a cross bar 47 of the base 26. A roller 48 mounted on the bracket 40 and running on the underside of the bar 47 prevents the bracket from rising. Due to the length of the links 41, the transverse movement of the magnet 39 permitted by limited swinging movement of the said links is nearly rectilinear.

The transverse movement is imparted to the bracket 40 through an upstanding post 49 secured to the bracket by side cheeks 50 and engaged by a roller 51 which is supported on a rotatable plate 52 eccentrically in relation to the axis of rotation, the post being held in contact with the roller by a tension spring 53. To permit of variation of the amount of the transverse movement produced by each half revolution of the plate 52, the roller 51 is mounted on an arm 54 connected to the plate by a pivot 55 at one end and adjustably at the other end by means of an arcuate slot 56 in the plate and a clamping screw 57 passing freely through the slot and engaged in the arm. In the position of the arm 54 shown in Figure 5 the transverse movement produced will be a maximum. With the arm 54 adjusted to the other end of the slot 56, the movement will be very small. The plate 52 is fast on a shaft 58 driven through worm gearing 59 from an electric motor 60.

Also fast on the shaft 58 is a cam disc 7% engaged under the influence of a tension spring '71 by a roller 72 on an arm 73 which is fast upon a shaft '74 extending to the other end of the base. Fast upon the remote end of this shaft is an arm 75 connected by a link 76 to a bell-crank lever 77 which carries a spring-loaded pawl 78 coacting with a ratchet wheel 79. The ratchet wheel is fast on the screw 35 and is wide enough to remain engageable by the pawl 78 in spite of the maximum possible longitudinal movement of the screw. As will be seen from Figure 5, the cam disc 70 has two notches 80 into which the roller can enter, thereby causing the pawl 78 to be rocked clockwise and to drive the ratchet wheel 79 and screw 35 forward. An adjustable pawllifter 81 is provided by which the effective movement of the pawl 78 can be limited to a fraction only of its actual rocking movement (i. e. one or more tooth pitches out of the number corresponding to the throw of the pawl). Return movement of the screw can be imparted by hand through a head 82 which may be provided with a scale, co-acting with fixed indicating means, similar to that of a micrometer gauge.

The shaft 58 may also drive a counter 83 through gearing 84.

The operation of the mechanical stage will now be briefly described.

Upon starting the motor 60, the shaft 58 turns the plate 52 which, in each half revolution, will drive the post 49 fully to the left in Figure 5 and allow it to be returned fully to the right by the spring 53. Figure 5 itself shows an intermediate position. As a result, a full transverse movement is imparted to the platform 22 through the magnet 39, the actual movement of the platform (i. e. the length of line scanned) being limited as required by adjustment of the stop screws 30. The movement of the post 49 and magnet 39 is set by the adjustment means 56, 57 to be slightly larger than is necessary to produce movement of the platform between the stops 30. Each transverse movement of the platform causes a line to be scanned on the slide 5. At the end of each transverse movement or line, the roller 72 enters a notch 80 in the disc 70 and the ratchet wheel 79 is turned by the pawl 78, causing the screw 35, the platforms 22 and 24 and slide 5 to be advanced longitudinally by an amount corresponding to the pitch between two lines. The pitch can be set by adjusting the pawl lifter 81. This sequence of operations continues automatically, the number of lines scanned being registered on the counter 83. When the full area to be scanned has been covered, the back of the stop plate 24' trips a micro-switch 85 in the circuit of the motor 60 and brings the device to rest.

To permit of focussing and adjustment and of visual examination of the image produced by the microscope 6, a mirror 86 (Figure 1) is provided which can be swung from a normal position (full lines) to a position 86 (chain lines) in which it reflects the beam from the semimirror 9 or Swan cube 9 on to a ground glass screen 87 furnished with a graticule. An eye-piece 88 is also provided at the middle of the screen.

In an alternative manner of carrying the invention into effect, a binocular microscope is used as a dividing element instead of the semi-mirror 9 or Swan cube 9, the arrangement being otherwise as described above.

As will be evident, it would also be possible to scan narrow zones at both marginal edges of the main zone and to arrange for half of the total number of particles detected in the marginal zones to be substracted from the number detected in the main zone.

If it is desired to discriminate for a given size of particle, it is only necessary to design or bias the discriminatory circuit 18 in well known manner so that it will respond only to a given amplitude of pulse from the photomultiplier 12. For obtaining a count of the number of particles in each of a number of size ranges, the single amplitude-discriminatory circuit 18 may be replaced by a plurality of such circuits each responsive only to a particular range of sizes and each associated with a separate counting means. The number of particles in each size range can then be estimated. Alternatively, a series of discriminatory circuits may be provided each responsive to particles of not less than a certain limit size and arranged in known manner, upon responding to a pulse, to block, in respect of that pulse, circuits with a lower limit.

I claim:

1. Apparatus for counting small particles, such for example as blood particles, comprising magnifying means for scanning a sample of the particles, a mask through which the sample is scanned over a known area and which has an aperture only slightly wider than the magnified image of the largest particle that it is desired to detect, a mask through which the sample is simultaneously scanned and which has an aperture Whose width is only a small fraction of the width of the firstnamed aperture, particle-image detecting means disposed beyond each of the apertures in the said masks and responsive to the appearance in respective apertures of magnified particle images, and counting means operatively connected to the detecting means for counting the difference between the number of images which appear in the large aperture and the number which appear in the small aperture during scanning.

2. Apparatus for counting small particles, such for example as blood particles, comprising a microscope, at stage for supporting a sample of the particles in proximity to the objective of the microscope, mechanical stageoperating means for imparting a scanning movement to the said stage, a constant-intensity light source arranged for directing light through the sample into the microscope, a light-dividing means arranged for receiving the light directed into the microscope, photo-multipliers arranged in the paths of the parts into which the light is so divided and adapted for detecting magnified particle images produced by the microscope, masks having apertures arranged in the paths between the light-dividing means and respective photo-multipliers, one aperture having a width slightly larger than the largest particle image to be detected and the other a width which is a small fraction only of the aforesaid width, pulse-amplitude discriminatory circuits connected to the outputs of respective photo-multipliers, pulse-counting means and a gate circuit connected between the said discriminatory circuits and the said counting means, the discriminatory circuit connected to the photo-multiplier associated with the wider-aperture being responsive for transmitting pulses corresponding to all particle images appearing in the said aperture during scanning while that connected to the other photo-multiplier is responsive for transmitting only pulses corresponding to particle images which completely obscure the narrower aperture and the gate circuit being operative for transmitting a pulse to the counting means when it receives a pulse from the former discriminatory circuit but no pulse from the latter discriminatory circuit.

3. Apparatus for counting small particles, such for example as blood particles, comprising a microscope with a mechanically operated sample-supporting stage adapted for being moved with a scanning motion in relation to the microscope, dividing means for directing the magnified microscope image along two difierent paths, imageresponsive photo-cells arranged in respective paths, masks having apertures disposed in front of respective cells, one aperture having a width only slightly larger than the magnified image of the largest particle to be detected and the other aperture having a width which is only a small fraction of the aforesaid width, the apertures being relatively oiiset so that the cell associated with the narrower aperture scans a narrow marginal zone immediately adjacent to a wider main zone scanned by the other cell, pulse-amplitude discriminatory circuits connected to the outputs of respective cells, pulse-counting means and a gate circuit connected between the said discriminatory circuits and the said counting means, the discriminatory circuit connected to the cell associated with the wider-aperture being responsive for transmitting pulses corresponding to all particle images appearing in the said aperture during scanning while that connected to the other cell is responsive for transmitting only pulses corresponding to particle images which completely obscure the narrower aperture and the gate circuit being operative for transmitting a pulse to the counting means when it receives a pulse from the former discriminatory circuit but no pulse from the latter discriminatory circuit.

4. Particle-counting apparatus according to claim 3, I

References Cited in the file of this patent UNITED STATES PATENTS 2,045,124 Cummins et al. June 23, 1936 2,436,262 Miller Feb. 17, 1948 2,791,150 Stevens May 7, 1957 2,791,377 Dell et a1. May 7, 1957 2,791,695 Bareford et al. May 7, 1957

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