US 3695762 A
Apparatus for the examination of a stream of material which is required to be presented to a sensor responsive to electromagnetic radiations that are modified by the presence of the material, the apparatus comprising means for moving the material past the sensor whilst supported on a thin flexible strip.
Description (OCR text may contain errors)
United States Patent 15] 3,695,762 Tylko et a]. [4 Oct. 3, 1972 DEVICE FOR DISPENSING 3,475,951 11/ 1969 Goetz ..250/222 M PARTICULATE MATERIAL UPON A 2,966,593 12/1960 Leimer ..250/219 MOVING SURFACE 2,429,331 10/1947 Sachtleben ..250/219 X  Inventors: Jozef Kazimierz Tylko; Charles 3,056,029 9/1962 Budniek ..250/237 Peter H y of 0/0 Develop Denoel ment Company Limited, 13, Park NTS Place, St. James, London S.W.1., FOREIGN PATE OR APPLICATIONS England 469,916' 8/1937 Great Britain ..250/219  led: April 1970 Primary Examiner-Walter Stolwein  Appl. No.: 28,277 Attorney-Townshend & Meserole Related US. Application Data 57] ABSTRACT  fggg giggz g gg Apparatus for the examination of a stream of material which is required to be presented to a sensor respon-  us Cl 356/36 250/219 FR 33 /3 4 sive to electromagnetic radiations that are modified by 51 Int. .1 ..G01'n 1/00 the Pmence the material the apparatus  Field of Search...250/2l9 DF, 209, 219 WE, 237 means the m l Past the Sens Whilst 6,250/219 TH, 219 QA, 222 PC; 356/163, 199, fuPPmed h flexlble 200, 209, 36, 38; 81/43; 33/34, 40; 118/401 V 7'  References Cited 3 Claims 6 Drawing Figures UNITED STATES PATENTS 3,192,796 7/1965 Peeps et a1 PATENTEDnm 3 I972 SHEET 1 BF 2 lDDDDDDEIClDDDD/D SDGDDEUCIDCIDEIDQD PATENTEU E 3 I972 SHEET 2 [IF 2 DEVICE FOR DISPENSING PARTICULATE MATERIAL UPON A MOVING SURFACE This application is a continuation of Ser. No. 703,140, filed Feb. 2, l968, now abandoned.
This invention relates to dispersions. It relates specifically to a method for providing a dispersion of a particulate material upon a substrate such that individual particles of the material are supported for examination of the particles.
Thus the invention may be used to disperse biological material such as a cytological specimen on the substrate but it may also be used to arrange a histological specimen (which could be a series of tissue slices that might be joined in a ribbon) along the supporting substrate.
When it is required to examine a particulate material for example by means of a microscope it is usual to first prepare a glass microscope slide upon a surface of which the material to be examined is spread in a thin layer. When particulate material is to be examined it is desirable for the material to be spread over an appreciable area of the slide so that overlapping of particles does not occur to a large extent and at high magnifications this area is much larger than will fit the field of view of the microscope. For full examination of the material on the slide it therefore becomes necessary for the slide to be moved backwards and forwards across the field of view so that eventually the whole of the material on the slide is examined.
The scanning of the whole area of the deposited material on the slide in this way is time consuming and small portions of the whole area may even be accidentally missed when carrying out the examination.
it would be preferable if the material for examination could be arranged in a linear fashion on the substrate in a path the width of which was about the same or was only a small multiple of the diameter of the field of view of the microscope. The use of the term microscope in the present context is intended to mean not only a conventional optical microscope but also whatever type of sensor is required to perceive a particular characteristic which is of interest in a dispersed particulate material. For some purposes only a low power magnification might suffice but in other circumstances high magnifications may be required.
According to one feature of the invention, apparatus for the examination of a stream of material which is required to be presented to a sensor responsive to electromagnetic radiations that are modified by the presence of the material comprises means for moving the material past the sensor whilst it is supported on a thin flexible strip. The thin flexible strip may be an uncoated safety film base. If desired, the film base may be of the subbed kind having been treated to improve its adhesion for a surface coating and give generally better surface properties. The film base may also be of a porous texture for particular applications. The film may be provided in a continuous length. The material to be examined may be fixed to the film by any of the suitable techniques used in laboratories a dry fixture method has been found to be particularly suitable.
According to a further feature of the invention, ap-
paratus for depositing a dispersion of a particulate material for microscopic examination comprises a feeder in the form of two blades between which a drop of the dispersion medium may be supported, the feeder being locatable adjacent support means for a continuous pathway such that a narrow line of the dispersion medium may be deposited on the pathway. The distance between the two blades of the feeder may be made adjustable so that the width of the line will be variable like a line drawn with a draughtsmans ruling pen. Apparatus may be included for adding more of the dispersion medium to the liquid in the feeder so that the amount of loss by deposition on the pathway may be replaced. This technique only applies when it is required to deposit a substantially continuous rather than a discontinuous trace. If the sample to be deposited is a specimen of cytological material the quantity held in the feeder will generally be sufficient for the purpose of the analysis.
The geometry of the deposition apparatus will generally be such that the stream of dispersion medium leaving the feeder will spread to a width of about 0.1 to 0.6 millimeters on the moving pathway. The concentration of particulate material in the dispersant medium will be such thatthe material will be arranged conveniently for its microscopic examination at a given rate of travel.
The dispersion medium may be volatile and it may include a suitable binder so that the specimen material will become fixed by the binder to the surface of the film as the medium evaporates. The medium may alter natively have some solvent effect on the material of the film so that the surface of the film will become softened by the dispersant. The medium might alternatively be non-volatile and could present for example a sticky surface to the specimen so that the specimen would tend to adhere to the film surface. The specimen material will then tend to become attached to the film surface or to sink therein and upon evaporation of the dispersant the specimen material will become fixed in the film when it becomes hardened again. One solvent that has been found to be particularly suitable for this purpose is acetone. An alternative solvent is a mixture of acetone and acetic acid.
The action of the feeder and the spreading and evaporation of the dispersion medium of the surface of the film base has been found to have with certain dispersions a self classifying effect on the specimen material as it becomes deposited from the suspension. The pattern of the specimen material as it is presented to the microscope is in the form of a narrow band which may be up to about 0.6 millimeters in width running along the length of the film base. The specimen material is arranged within this band such that the larger particles of the specimen are positioned towards the center of the band and the smaller particles are positioned towards the outside edges of the band. This classification has been found to facilitate the microscopic examination of the specimen material on this band. For instance, if a fairly wide band of specimen is used it may be convenient to use one microscope to examine one edge of the band, another to examine the center and yet another to cover the other outside edge of the band.
Where the specimen material to be examined by the microscope is for example of biological interest it may be desirable for the specimen to be stained as is usual to bring out particular features of the specimen. This staining of the specimen may be carried out before the suspension of the specimen for coating on the film base is prepared. It is convenient however if the staining is undertaken after deposition on the base. The processes required for staining may comprise the treatment of the specimen material in a number of staining baths for critical intervals of time and this may readily be undertaken if the base is passed through these baths in sequence in a manner similar to that in which cinema film is processed. Staining is mentioned only as an ex ample and the specimen might be given any other suitable treatment. Thus the specimen can be preserved from any deterioration, i.e. autolysis and contamination by microorganisms by the use of suitable preservatives or medical fixatives.
The deposition of the specimen material on the film base may be effected in such a way that the particulate material contained therein undergoes segregation according to its size, weight and hydrodynamic resistance so that a trace in which the particles exhibit substantial segregation with respect to a vertical axis is obtained. A characteristic distribution may be obtained which can show a bimodal mode of distribution of particles with respect to the longitudinal axis.
Dispersion of the specimen into its primary particles to aid track laying and observation of the specimen may be facilitated by mechanical means such as ultrasound and viscous shearby chelating agents such as ethylene-diamine-tetra-acetate citrates and oxalates by mucus dispersants such as urea, thiourea, sodium chloride, acetyl cystein, glutathione, ascorbic acid, potassium and sodium metabisulphite, by enzymes such as trypsin, papain, streptokinase, streptodornase and hyaluronidase or mixtures thereof by hormones such as oestrogen andsubstances having an oestrogen like action by alcohols such as glycerol and glycol by polymers such as polyvinylpyrrolidone by surface active agents and by the control of pH, ionic strength, and buffer content of the specimen.
Preferably, the trace containing the cells may be sealed with a varnish such as a methacrylate based coating preparation, i.e. as sold under the proprietary name of Bedacryl [22X or a glyptal based proprietary varnish such as Beckosol P.470. Alternatively, various types of commercial nail varnish may be used.
The biological staining before the specimen is deposited on the film base may be effected by one or more stains the purpose of which is to provide a differential staining of various components contained within the particulate material.
Staining of the specimen prior to laying of the trace may be accomplished by addition of a known quantity of stain to the specimen with adjustment of the pH of the specimen. As an example a specimen of cervical cells obtained by a cervical scrape can be placed in lml. of water containing 0.1 percent w/v methyl phydroxy-benzoate, 0.2 percent w/v potassium metabisulphite, 0.1 percent methylene blue adjusted to pH 8.0 with sodium hydroxide, left for a period in excess of 2 hours and then used to form the trace.
For instance the method may also be used to introduce one stain which produces one color or range of colors within the visible spectrum in particular regions of the particulate material, together with another stain of the fluorochrome type, which, when excited by a suitable ultraviolet radiation, will emit a different color in the visible spectrum.
Staining of the specimen may be carried out after laying on the film base as will be described later by any method known to medical science.
Facilities for optically examining the specimen of biological material on the film base may be provided in line with the apparatus for depositing the trace on the film or these facilities may be provided separately. The examination may be effected by means of transmitted or reflected light, dark ground illumination, phase contrast microscopy, transmitted or absorbed infra-red, radiation, or U.V. radiations in such a way that the whole width of the trace lies within the field of view of the means for making the examination.
The invention also comprises a method and apparatus for laying upon a surface of a film base a ribbon or series of laterally adhering sections derived from a histological sample sliced by a microtome.
The surface of the film base after the trace containing the specimen has been deposited thereon may be coated with a thin layer of a transparent and suitable sealing material which will seal over the whole width of the deposited specimen. After being dried under controlled conditions the sealing material will provide a substantially flat and smooth surface with the specimen embedded therein. The combination of film base, specimen and sealing material is then flexible enough to permit it to be transferred to a standard film reel for storage of the base and specimen.
By way of example, embodiments of the invention will be further described with reference to the accompanying drawings in which,
FIG. 1 is a perspective view of a microscope stage according to the invention with a microscope in position,
FIG. 2 shows a portion of a flexible film base carrying a dispersion of particulate material for microscopic examination.
FIG. 3 shows a system in which a specimen may be examined successively by each of three microscopes, and
FIG. 4 shows in greater detail a view of a feeder of the non-disposable type for a dispersion of particulate material taken from the direction of the arrow 17 in FIG. 3.
FIG. 5 shows a disposable container with labels of the type particularly useful for the storage, transport and treatment of cytological material, engaged in a feeder.
FIG. 6 shows a feeder of the disposable type.
FIG. 1 shows a microscope stage 1 which is supported between a microscope 2 and an illuminating lamp 3. The microscope stage 1- comprises a shallow trough 4 in which on the optical axis of the microscope 2 is a window 5. The trough 4 is of such width that a strip of 35'millimeter film base will just lie therein and be located in the trough by sidewalls thereof.
A strip of film base 6 is shown in FIG. 2 and this carries the usual sprocket holes 7. The strip in the Figure has had deposited on'it by the method about to be described a thin trace 8 of a dispersion of a particulate material which is to be examined by the microscope 2. The film base 6 also carries a magnetic stripe 9 the use of which will be explained later. When the film base 6 is fitted into the trough 4 of the microscope stage 1 the position of the window 5 is such that light from the lamp 3 will be able to pass through a small length of the trace 8 and into the microscope.
The method of depositing the trace 8 of particulate material on the film base 6 is best seen from FIG. 3. The drawing shows a length of film base 6 which is fed from a feed spool 10 over a free running sprocket roller 11 and across a microscope stage 12. In this example the microscope stage 12 serves as a stage for three microscopes 13 which are mounted side-by-side above the stage 12. A given small length of the trace 8 will thus pass beneath each of the microscopes 13 in turn and these microscopes will have been adjusted appropriately so as to observe a different characteristic of the particulate material in that portion of the trace 8. Each microscope may be arranged to view the whole width of the trace and the observations may be carried out with different wavelengths of light. The use of a wavelength of electromagnetic radiation not in the visible spectrum is also envisaged.
After passing across the microscope stage 12 the film base 6 crosses a further free running sprocket roller 14 and then is wound on to a take-up spool 15. Drive means, not shown in the drawings, are included to enable the film base 6 to be driven at a constant speed from the feed spool to the take-up spool. The drive means also enables the base to be inched forward and to be driven fast in a forward or reverse direction. As the film base 6 passes over the roller 11 a dispersion of particulate material for examination by the microscope is applied to the base to form the thin trace 8. The application of the dispersion to the base is effected by means of a non disposable feeder 16. This type of feeder is shown in greater detail in FIG. 4 which is a view from the direction of the arrow 17 in FIG. 3 on a greatly enlarged scale.
The feeder 16 comprises two blades 18 which are joined together at a common end but are slightly separated at a tip end 19. The distance between the tips of the blades may be varied by adjustment of a screw 20 which is fitted between the two blades. The blades are mounted on an arm 21, which is fixed relative to the position of the roller 11 carrying the film base 6. The space between the tip ends 19 of the blades is suitable for holding a small volume 22 of dispersion medium in which particulate material 23 to be deposited on the base 6 is suspended. When the base 6 is being driven through the apparatus, the tip ends 19 of the blades are positioned very close to the base but do not actually make contact therewith. A flow of the dispersant medium from the volume 22 between the blades is started by touching the tips of the blades and the base 6 with a drop of the dispersant medium on a glass rod. The glass rod may then be removed and the flow of the medium from the feeder will then continue and will result in a thin trace of the medium being deposited on the moving length of the base 6. The particulate material 23 carried in the medium will become transferred to the surface of the base 6 and will become dispersed thereon in an arrangement making them suitable for examination by the microscope 13.
A preferred mounting position for the feeder 16 is on the same horizontal line as the axis of the roller 11. Continued operation of the feeder will cause depletion of the volume 22 of dispersant held therein. A capillary tube 24 linked to a reservoir is thus arranged to feed further quantities of the dispersant medium to the feeder so that the amount of liquid held in the feeder remains substantially constant.
In operation it is found that the trace of medium which is deposited on the base 6 can be held to a sufl*"- ciently constant width and the particles of material carried in the medium are deposited in a suitably linear fashion. The dispersant medium between the blades remains in a state of constant agitation while the apparatus is in operation and there is little tendency for sedimentation of the suspended material to occur at this point. However, for some applications it is desirable for segregation of suspended particles due to sedimentation to be retarded and this may be done by vibrating or stirring the volume of liquid in the feeder. For instance the blades 18 of the feeder may be vibrated by placing them in an alternating magnetic field.
FIGS. 5& 6 shows a disposable container with the attached disposable feeder and a disposable feeder respectively. The flexible and pliable container 25 is integrally joined by means of the strap 26 with the bung 27. The bung carries a flexible reversible collar 28, which is reversed after the closure of the container. The lower end of the container is in the form of a funnel 29. The tip of this funnel has been cut away prior to the engagement in the feeder duct. By gentle squeezing of the container 25 the liquid with the suspended particles descends into the feeder 31 and reaches its protruding tip 32 through which it ultimately emerges in the form of a narrow trace containing the suspended particles. The passage 33, the protrusion 34 and the recess 35 are all used for the purpose of location and manipulation of the feeder.
The dispersant medium after being used to convey the suspension of particulate material for examination to the film base and arrange it thereon will generally be allowed to volatilize so that the particulate material will be supported by the film base in direct contact with the base and in a substantially dry condition for the microscope examination. The particulate material will tend to adhere to the film base and this adhesion may be improved by appropriate treatment of the base prior to deposition of the material. Subsequent coating as described earlier, may be applied when necessary.
By way of example a use of the dispersion method of the invention will be described in a medical application involving the examination of cytological smears.
A sample of exfoliated cells collected by Ayres spatula from the region of the squamo-columnar junction of the cervix was suspended in acetone and introduced into a capillary tube 24 in which the suspension was vibrated with a small micro-vibrator operating at 50c/s. From the capillary tube it was dosed at about 1 milliliter per 30 seconds into the volume 22 between the blades of the feeder. The film base 6 was a 35mm cellulose acetate film which was driven through the apparatus at lOcm/sec. The feeder deposited the suspension of cells on the base in a thin trace 8 which was 032mm wide with a width tolerance of 1004mm. Three portions of the prepared dispersion on the film base 6 were stained by the Papanicolaou, shortened Papanicolaou, and Shorr methods respectively. When examined under an optical microscope after evaporation of the acetone and drying of the cells on the base these portions showed good staining, a complete absence of clumping of the cells and a generally good readability.
The film base carrying the dispersion of cells formed in this way was stored in a wound up condition on a roller of inches diameter for 7 days and then repeatedly rewound over a 19mm diameter roller after this treatment the adhesion of the dispersion to the base was still good and no cracking or peeling off of the deposit was observed.
it has been found that an improvement can be effected if the trace on the film base is sealed after it has been laid down by some means such as applying a thin coating of a lacquer or varnishes preferably of the types mentioned before. The effect of this is to smooth out irregularities in the trace on the film, to strengthen the trace and help to prevent accidental peeling of the trace from the film. Sealing also facilitates the use of an immersion microscopy technique if this is required for repeated examination of the specimen.
The magnetic stripe 9 on the film base 6 is intended to be used in conjunction with magnetic recording and reproducing heads (not shown in the drawing) which are mounted in the dispersion apparatus alongside the path along which the film base is moved. The sprocket holes 7 on the film base could be used to give a rough indication of the position of any point on the thin trace 8 carrying the dispersion that was of particular interest but a more accurate location can be indicated by means of an appropraite signal recorded on the stripe 9.
The same or a separate magnetic stripe on the film base may be used to record information on the base in a predetermined position with respect to a corresponding particle of material under examination. An observer using one of the microscopes 13 may thus cause a signal to be recorded on the base when he detects a particular characteristic in a particle of material under observation in the apparatus. Alternatively, a mechani cal observer of the characteristic may be caused to record this information. Information stored in this way on the base may be used possibly in conjunction with other information from a suitable store, to adjust or condition later microscopes through whose fields the film base will pass for making selected observations of characteristics of the dispersed particulate material.
The foregoing description of an embodiment of the invention has been given by way of example only and a number of modifications may be made without departing from the scope of the invention. For instance, instead of using the capillary tube 24 for maintaining the volume 22 of dispersant medium in the feeder it would be possible to use a suitable pump to add this liquid. The example was particularly directed to apparatus for the microscopic examination and evaluation of cervical smears but it is clear that it could be employed in other medical applications such as in exfoliative cytology,
haematology, chromosome studies or bacteriology. When the apparatus is to be used for cytological analysis, to reduce the likelihood of contamination between samples it has been found convenient to use the feeder only once .for each sample in which case it may be made in disposable form. A feeder constructed of a plastics material as described above has been found to be suitable and is capable of being produced at a low cost. The provision of a linear dispersion of particulate material upon a substrate is also useful in the examination of fine powders, such as pigment, cement, r netal powders for a particle size analysis or the recognition 0 crystal or bacteria shapes and in other applications for industrial and research purposes.
1. In a device for dispersing a substantially symmetrical arrangement of discrete particles in substantially non-overlapping relationship on a moving surface for subsequent microscopic examination the combination comprising, a chassis, means supporting a length of film base constituting a substrate on said chassis, drive means connected to said support means to move said substrate relative to a fixed point on said chassis, feeder means mounted on said chassis and positioned to selectively deposit a liquid dispersion of said discrete particles in substantially non-overlapping relationship in a symmetrical narrow trace of substantially constant width with said discrete particles symmetrically distributed about a longitudinal axis of the trace with respect to their size on said substrate, said feeder means including a body portion and a blade portion, said blade portion defined by a pair of spaced blades of equal length separated by an elongated opening extending substantially into the body and dimensioned to receive a quantity of said liquid dispersion therewithin for delivery to said substrate, said blade portion further including means to vary the width of said opening by moving said blades with respect to each other, one end of said blade portion disposed out of contact with but in sufficiently close proximity to said substrate to deposit thereon the liquid dispersion disposed in said opening of said body in a controlled trace of substantially constant width and depth and supply means associated with said feeder means to deliver a constant supply of liquid dispersion to said feeder for delivery to said substrate.
2. The apparatus of claim 1 further including viewing means for optical examination of said trace on said substrate.
3. The apparatus of claim 2 wherein said chassis includes means defining a slot beneath said substrate adjacent said viewing means and a light source directed at said slot to illuminate said substrate.