|Publication number||US3572890 A|
|Publication date||Mar 30, 1971|
|Filing date||Nov 18, 1968|
|Priority date||Nov 18, 1968|
|Publication number||US 3572890 A, US 3572890A, US-A-3572890, US3572890 A, US3572890A|
|Inventors||Adamik Emil R|
|Original Assignee||Atomic Energy Commission|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (5), Non-Patent Citations (1), Referenced by (12), Classifications (8)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent  Inventor Emil R. Adamik 3,204,523 9/1965 350/67X Laurel, NY. 1,198,402 9/1916 269/21X  Appl. No. 776,736 2,300,252 10/1942 350/94  Filed Nov. 18, 1968 2,825,261 3/1958 350/95X  Patented Mar. 30, 1971 OTHER REFERENCES  Assigns: g f i s i gz ggzgz g: Masterson, Vacuum Spintable, l B M TECHNICAL gy DISCLOSURE Bulletin" Vol. 5, No. 5, Oct. 1962, pp 7 8.
ommlsslon Primary Examiner-David Schonberg, Assistant ExaminerT. H. Kusmer  MICROSCOPE SLIDE AND SPINNER Atmmey R0|and Anderson 6 Claims, 2 Drawing Figs.
 US. Cl 350/92, 356/246 [51 Int. Cl G02b 21/34 ABSTRACT; M a fo examining biological cells of the type Fleld of Search deposited from ara and used in blood| cel] studies comprising --95; 356/244, 246; 248/362; 269/21, 57 a slide having barrier means supported on a rotatable platform formed with a flat depressed portion for receiving, supporting  References cued and rotating the slide to produce a monolayer of cells confined UNITED STATES PATENTS on the slide by the barrier means for examination by conven- 1,918,351 7/1933 Schulze 350/94 tional microscopy, as well as automatically.
\\\\t\ 2 3 I 5 l 3 2 7 vAcuu M PUMP PATENTEU man 1971 MOTOR aim" F/GZ INVENTOR.
EMIL R. ADAMIK MICROSCOPE SLIDE AND SPINNER This invention relates to the field of biology and more particularly to the microscopic and autoradiographic examination of biological cells.
2. Description of the Prior Art Conventionally, the examination of biological cells involves the production of cell smears. To this end a small drop of cellcontaining material, such as sera containing blood cells, is placed on a microscope slide and spread thereon by squeezing the sera between the slide and a thin glass cover slip. Altemately, the cover slip is drawn across the drop of cellcontaining material to smear the sample across the slide. On particular disadvantage of these methods and apparatus is that the smears are not uniform. This is due to the human errors inherent in the manual manipulation of the cover slips. Also, the cell samples themselves are taken from a wide variety of human and nonhuman subjects resulting in wide differences in cell samples, differences in cell types, numbers and density and or in variations in the viscosity of the sample. Thus, it is extremely difficult, impossible or time consuming and expensive to produce uniform, monolayers of cells by conventional techniques. Also, the cells are often broken, disrupted or otherwise damaged due to the forces required in making the smear with conventional cover slips. For example, grossly disrupted, distorted or degenerated cells often make up more than percent of all leukocytes encountered on scanning conventional coverglass smears. In this regard, large lymphocytes, with abundant cytoplasm and noncompact nuclei, are particularly subject to damage. Moreover, the cells often involve radioactive material and it is difficult to handle these materials with conventional methods and apparatus. It is additionally desirable to provide an automatic or semiautomatic system for the production of a monolayer of cells for microscopic, automatic and/or autoradiographic examination.
BRIEF SUMMARY OF THE INVENTION The invention described herein was made in the course of, or under a contract with the US Atomic Energy Commission.
In accordance with this invention a sample containing biological cells is applied to a glass microscope slide having barrier means thereon for confining the sample as the slide and sample are rapidly rotated. More particularly, in one embodiment a slide having grooves thereon is assembled in a holder adapted to be rotated by a chuck capable of pulling a vacuum therethrough whereupon the slide and holder as sembly are rotated rapidly and the grooves on the slide impede the flow of excess material across the slide due to centrifugal force thereby to produce a uniform monolayer of cells on the slide. With the proper selection of components and their arrangement, as described in more detail hereinafter, the desired uniform monolayer of substantially undamaged cells is achieved for examination with good cytological detail.
The above and further novel features and objects will appear more fully from the following detailed description when the same is read in connection with the accompanying drawings. It is to be expressly understood, however, that the drawings are not intended as a definition of the invention but are for the purpose of illustration only.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING In the drawings, where like elements are referenced alike:
FIG. 1 is a partial three-dimensional view of one embodiment of the spinner and slide assembly of this invention; and
FIG. 2 is a partial cross section of a practical system for producing a monolayer of cells with the spinner and slide assembly oflFIG. l1.
DETAILED DESCRIPTION While the apparatus and method of this invention are par ticularly useful in the autoradiographic examination of whole blood samples from a wide variety of human subjects and nonhuman subjects, such as dogs, cats, calves, birds, etc., it will be understood that this invention is also useful in microscopic or automatic examination of a wide variety of radioactivity or nonradioactivity containing blood or other cell-containing liquid biological samples where uniform cell distribution is advantageous. In this regard, the cells may contain tritiated thymidine H-TdR), whereby the percentage of radioactively labeled blood cells can be counted by the use of autoradiographic photomicrographs, planimetric measurements, or automatically by inserting the samples in plastic test tubes for scintillation counting with thallium activated NaI crystals. Also, the cells may be stained with Wrights or other stains, be unstained, contain large or small numbers of leukocytes, erythrocytes, or other cells, or be undiluted or diluted with various materials such as EDTA anticoagulents, autologous serum, isotonic phosphate buffered saline (pH 7.4, 300""sm/L), 6 percent bovine serumalbumen, or radioiodinated human I-serum albumin.
As is known autoradiography is one of the most commonly used techniques in cell and tissue biology, It has widespread applications ranging from the delicate study of a replicating DNA molecule in bacterial cells to localization of metallic products in different parts of a mammalian tissue or organ. In cancer research, autoradiography is particularly useful in studying metabolic activities of cells and cellular components and kinetics of cell proliferation and differentiation. Briefly, the process of radiography is based upon the same principles as light photography. Autoradiography, however uses ionizing charged particles, such as a and B particles emitted by disintegrating radioactive atoms. Thus, the a and B particles produce changes in the silver halide crystals in the photographic emulsions like those produced by the conventional light photons and the latent image is transformed into a visible image by developing and fixing. As will be understood in more detail hereinafter, this invention is particularly useful in producing a monolayer cell sample where the distance between the actual locus in the sample and the visible image is only of the order of a few microns or less, as it is when the sample is in direct contact with the sensitized photographic emulsion.
Referring now to FIG. I, the apparatus 11 of this invention, comprises a support member I3 forming a tubular sleeve 15 around a right circular, cylindrical opening I7 having an O- ring I9 disposed therein in an O-ring groove around the axis of the opening 17. Platform 2! extends from around the circular opening in cylindrical sleeve 15 normally to the axis of the opening I7 and forms a flat depressed portion 23 also extending longitudinally at right angles to the axis of opening 17 and extending longitudinally from around the opening I7. Ad vantageously, the support 13 is made from clear plastic, such as acrylic-molding resin which is easy to form, machine or cut. To this end, the sleeve 15, the groove machine O-ring I9 and opening 17 are turned and/or bored by conventional means, the platform 21 is cut by conventional means, and the depressed portion 23'is milled by conventional means. Altemately, the support may be molded in one piece and finished to size by conventional machining.
Glass slide 25 fits snugly in depressed portion 23 to form an integral assembly II with support member 13. This slide 25 has a rectangular plate shape forming a first, flat, side 27 extending longitudinally along the flat bottom of depressed portion 23 of support member 13. A second major side 29, which is opposite and parallel to the first side 27 of slide 25, has a barrier means 31 thereon for receiving and confining cell-containing sera and/or photographic emulsion when the assembled apparatus 11 of this invention is rotated. Advantageousiy, the slide is l-inch wide by 3 inches long and barrier means 31, comprises two narrow grooves scored at l-inch intervals For example, two grooves having shallow depths are cut with a diamond point at l-inch intervals to provide three l-inch X 1- inch divisions. Altemately, the grooves are ground in intaglio transverse to the long rectangular dimension of slide 25 to provide the two grooves. Typical grooves are 0.0l-inch wide and 0.010-inch deep.
Advantageously, a central inch square area is provided between the two grooves and the ungrooved edges of side 29 intersecting minor sides 33 of slide 25 and this central inch square area has a center coinciding with the axis of opening 17 to form a surface at right angies to this axis of opening 17. Also, the slide 25 has a thickness of at least 0.040 inches. This thickness prevents breakage from handling and it permits the transverse grooves to be scratched or ground therein without breakage during the application of the grooves or the later handling of the slide. Additionally, this thickness prevents deflection of the center of the slide 25 when a vacuum is applied to one side thereof as described in more detail hereinafter.
in operation, minor sides 33 of slide 25 advantageously extend into the depressed portion 23 of support member 13 at right angles to the opposite major sides 27 and 29 of slide 25, as shown in MG. 2. A drop of the cell-containing sample, such as whole blood, is spread with a blunt instrument, such as the end of an eye dropper, over the inch square area at the center of slide 25 between the two opposing grooves forming barriers 31 on side 29 thereof. A rotatable shaft, forming a chuck 35 having internal holes 37 coaxial with the axis of the chuck, rotates the support member and slide 25 in assembly 11 to form the desired monolayer 39 on side 29 of slide 25. To this end, motor 41 rotates the chuck at 5000 r.p.m. while vacuum pump 43 pulls a vacuum of above inches of Hg through holes 37 in chuck 35 to hold the slide 25 in sealing contact against the flat bottom of depressed portion 23 of support member 13 and to hold the outside of chuck 35 in sealing contact with the elastic O-ring 19 and in close fitting contact with the inside diameter of sleeve 15, which forms opening 17. Thus, even if motor 41 brings the support member to full speed in a short time, such as 30 msec. to 1 see, the support member 13 has a strong positive but selectively removable vacuum connecting force for coupling the support member 13 to the chuck 35 to prevent relative slippage therebetween. Also, this force on the support member 13 and slide 25 closely and strongly couple these two elements of assembly 11 together substantially without deflection of the center of the slide 25 and substantially without relative slippage therebetween that would prevent getting the support 13 to full speed quickly.
The rotation of assembly 11 at 5000 r.p.m. for about 15 seconds produces a dried monolayer 39 of cells at the center square inch area of slide 25 and removes the excess material from the side 29 of slide 35. To this end, the combination of the surface, tension of the cells and, the resistance to and/or the flow of the cells, sera, etc., created by the barrier 31, provides the necessary resistance to and/or the flow of the excess material through and/or across the barrier 31 on the slide. Thus the desired uniform density of cells is confined to the center inch square area of the slide 25 and a uniform distribution of dried undamaged cells is produced in monolayer 39. This is significant since a high rotational speed is required quickly. Thus, without the barrier 31 the imposed centrifugal force necessary to produce the monolayer, e.g. 5000 r.p.m. imposed rapidly and lasting for about 15 seconds, causes streaking of the cells, sera, etc., that make up the sample drop placed on the slide 25.
Advantageously, the top surface 29 of the slide 25 is above the top surface of support member 13, which is above and all around the flat bottom of its depressed portion 23, whereby the excess material from the blood sample is slung off the slide 25 by the high centrifugal force involved and over and above the support member 13 without hitting the support member 13 or lodging in the depressed portion 23. Thus, no radioactive material is caught in or on the support member 13 and a cover, not shown, may be placed over and around the rotating assembly 11 to catch the excess material and to shield the operator from being sprayed with excess radioactivity containing material therein.
in actual operation, the apparatus and method of this invention produces a uniform monolayer 39 with substantially little or no damage to the cells in the monolayer. For example, dried monolayers 39 are routinely and repeatedly produced at 5000 r.p.m. with flattened undamaged cells. In this regard, lower speeds, such as 3000 r.p.m., produce a significant lack of uniform cell density and high speeds, such as 9000 r.p.m., produce significant cell damage. Moreover, greater cell density in the central inch square area of slide 25 due to dishing of the slide is prevented since the thickness of the slide 25 prevents the one side thereof from causing deflection of the central inch square area of the slide.
After applying a cellular monolayer 39 to slide 25, as described above, a photoemulsion is applied thereto for autoradiographic purposes. To this end motor 41 and vacuum pump 43 shut off to release slide 25, the slide is dipped in a conventional emulsion, the slide is reinserted in depressed portion 34, the vacuum pump 43 reapplies a vacuum to slide 43, and motor 41 rotates the assembly 11 of support member 13 and slide 25. This produces a desired 2-5p, thick layer of photographic emulsion on cell monolayer 39.
As shown in FIG. 2 characteristic developed silver grains 45 are produced above the radioactivity in the nucleus and cytoplasm of the single cell beneath gains 45, and characteristic developed silver grains 47 are produced above radioactivity in the nucleus of the cell beneath grains 47, while cell 49 is clearly unlabeled. For ease of explanation, the size of the cells in monolayer 39, such as cell 49, have been drawn larger than scale to illustrate that the cells in monolayer 39 must be uniformly distributed therein to a one cell depth an a thin emulsion layer 51 must be uniformly coated directly thereon in intimate contact therewith.
As understood from the above, the photographic emulsion layer 51, comprises a dispersion of silver halide crystals under a transparent gelatin layer 53. Slight imperfections in the crystal lattices are purposely introduced during the manufacture of the silver halide. When energy from light photons or ionizing particles is dissipated within the emulsion, electrons migrate and concentrate at the sites of the slight imperfections, which form electron traps. The presence of these electrons renders these sites negatively charged, and this in turn causes positively charged silver atoms in the vicinity to migrate to the sites, neutralizing the charge and producing silver atoms. The process repeats until a latent image, an aggregate of several reduced silver atoms, is formed at the imperfection site. The process of latent image formation is accomplished by the liberation of brownie from the crystal, so that the latent image comprises the metallic silver grains shown in FIG. 2.
While the process of this invention has been described with reference to a single autoradiographic emulsion layer it is understood from the above that the process of this invention can likewise be used to produce a lower emulsion layer for producing developed silver grains from H only and an upper emulsion layer separated from the bottom layer by a celloidin layer. In this case, the upper layer is used to produce developed silver grains from 'C only and/or 11 and C. To this end, a celloidin and an upper emulsion layer are produced by respective rotations of the assembly 11 as described above with reference to the single emulsion layer after application of the respective compositions of these layers. in each case the slide 25 is removed from support member 13 by inserting a pointed tool in cut out portion 55 in support member 13 at the corners of depressed portion 23.
It is also understood from the above that this invention is aiso useful in producing a uniform monolayer of undamaged biological cells for conventional microscopic or planimetric examination.
This invention has the advantage of safely, effectively, efficiently and reproducably producing a uniform monolayer of biological undamaged cells for autoradiographic, microscopic or planimetric examinations. Moreover, the apparatus of this invention is easy and inexpensive to make and use, and provides positive unambiguous identification of the side of the slide upon which the monolayer is produced.
1. Apparatus for examining biological cells comprising:
a. a vacuum spinner having a shaft forming a passage therethrough for drawing a vacuum through said passage while rotating said shaft at high speed;
b. means forming a support for rotation with said shaft having a platform forming a sleeve around an annulus for pulling a vacuum through said passage and platform while said support is being rotated by said shaft, and
a flat, rectangular, plate-shaped slide having a first, major, flat surface for rotation by said support by pulling said vacuum against said surface while said surface is in contact with said platform, and a second, major, flat surface parallel with said first surface forming two, spaced-apart, uniform cross section, open ended grooves, extending continuously and uninterruptedly across said second, major, flat surface whereby a suspension containing biological cells can be placed on said second, major, flat surface between said grooves for rotation of said slide by said vacuum spinner and support for communicating said suspension with said grooves for separating a uniform, substantially undamaged monolayer of said cells from said suspension and depositing said separated cells on said slide between said grooves.
2. The invention of claim 1 in which said platform forms below a shoulder a depressed area whose depth is less than the thickness of said slide, which corresponds to the distance between said firstand second, major, flat surfaces, whereby cells containing radioactive material can be applied to said slide and an excess thereof can be removed from said slide by centrifugal force during the rotation of said slide by carrying said excess over said shoulder so as to prevent the accumulation of radioactivity containing material on said support, said shoulder and in said depressed area.
3v The invention of claim 1 in which said slide is a l-inch X 3-inch X 0.040-inch glass, rectangular, plate-shaped, microscope slide, said grooves are two l-inch long, grooves 0.010-inch wide by 0.0l0-inch deep below the surface of said first, major flat surface and uniformly 1-inch apart for receiving said cell-containing suspension in an inch square area between said grooves, whereby a flow of said cell-containing suspension along said first, major, flat surface is produced by the rotation of said slide and is confined for transportation by said grooves for the uniform separation distribution and deposit of said monolayer of said cells on said slide between said grooves during said rotation without substantial deflec' tion of said slide by said vacuum.
4. The apparatus of claim 1 for the preparation of biological cells of the type deposited from sera having different viscosities and cell sizes used in microscopic and autoradiographic blood-cell studies, wherein said grooves are formed intaglio at equal angles to long edges on said first, major, flat, side to provide an inch square flat area the center of which is supported by said support coaxial with the axis of rotation of said shaft for receiving and rotating said slide and cell-containing suspension applied to said slide between said grooves at right angles to said axis to substantial said monolayer of cells on said slide form microscopic as well as automatic examination of said cells. v
5. The apparatus of claim l'in which said support has raised shoulder means around the perimeter of said slide for holding said slide against lateral movement during said rotation, and for centering said slide symmetrically relative to the axis of rotation of said shaft.
6. The apparatus of claim 1 in which said support has O-ring means in said sleeve for providing vacuum sealing contact with said shaft for providing for the selective insertion of said shaft into said sleeve for attaining a slide rotation speed of 5000 r.p.m quickly without relative slippage between said shaft and sleeve.
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|Citing Patent||Filing date||Publication date||Applicant||Title|
|US3827805 *||May 24, 1973||Aug 6, 1974||Corning Glass Works||System for controlling centrifugal forces to produce cellular monolayers|
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|U.S. Classification||435/283.1, 359/396, 427/2.11, 422/72, 356/246|