US 3526203 A
Description (OCR text may contain errors)
United States Patent  Inventors Thomas D. Kinney 3120 Devon Road, Durham, North Carolina 27707; John E. P. Pickett, 2323 Pinafore Drive, Durham, North Carolina 27705  Appl. No. 719,434  Filed April 8, 1968  Patented Sept. 1, 1970  ELECTRON MICROSCOPY TISSUE PROCESSOR 5 Claims, 10 Drawing Figs.
 U.S.Cl 118/7, 118/429, 118/50, 118/500  1nt.C1 ..B05c 11/10, B05c 3/02  Field ofSearch 115/7, 50, 429; 134/95, 96; 1 18/500  References Cited UNITED STATES PATENTS 1,986,319 1/1935 Bongrand et a1 118/50UX 2,120,816 6/1938 Schnoll ll8/5OX 2,925,820 2/1960 Weiskopfet a1, 118/429X Primary Examiner-John P. McIntosh Attorney-B. B. Olive I ABSTRACT: A tissue processor for fixation, dehydration and clearing of minute, i,e. electron microscopy size, particles of tissue utilizes a porous receptacle for each particle of tissue and a processing chamber adapted to contain a plurality of the porous receptacles. The processing chamber is connected to various tanks which contain the usual aqueous and organic non-aqueous processing solutions. Fluid controls are provided so that a predetermined volume ofeach solution is successively brought to, retained in and drained from the chamber. The receptacle pores are such that each aqueous and non-aqueous solution is permeable through at least some of the pores thus allowing the particles to be bathed in the various solutions in any of several programmed time sequences and while residing within their respective porous receptacles throughout whatever time is required to complete the particular program.
91, 108 106 POWER 89 SIGNAL SELECTORlSWITCH TIME PROGRAM SUPPLY LIGHT MANUAL AUTDMAUC DELAY SWITCH 90 I Y CLOCK PROCESSlNG SOLUTION CONTAINERS l 2 i Q Z 5 9 LO I l 1 1 1 RI RIHIRRT WTT IL Patented Sept. 1, 1970 Sheet FIG. 4
L g g 5 5 $13 .5 E2 1% l v i l l 12] 0 92a 95a POWER 108 1 so -0 3E31 96 azaa m o 2 93/593) 199a WARNING 1O7 DELAY TIMER Z WAOLQ Low HIGH ST'RT 97g: kicqooa LEVEL LEVEL 196 97 T 401 :AN AuToerwlc 99 10 O 24 1 89 OFF MANUALQ PROGRAM PTY F L I INVENTORS Thomas D. Kinney FIG. 1
ATTORNEY Patented Sept. 1, 1970 3,526,203
47 INVENTORS I Thomas D. Kinney 47 5 BY John E. P Pickett FIG-2 ATTORNEY Patented Sept. 1, 1970 1 3,526,203
Sheet 3 of3 l 2 3. 4. E E z E a L l r l I I @4355 mgbyggisg: EggdggkZZ MANUAL IQ J VALVE t l 1 91 108 I06 POWER g; SIGNAL SELECTOR I SWITCH TIME PROGR SUPPLY LIGHT DELAY SWITC MANUAL 7 zAUTOMATIC CLQCK I PROCESSING SOLUTION CON TA I N E R S CONTROL TO PROCESSING CHAMBER S|GNAL SWITCHES LIGHT LEVEL PROCESSING CHAMBER CONTROL 81 80 I32 VACUUM WASTE TANK LEy'EL WARNING PUMP CONTROL LIGHT /I33 131 To WASTE FIG. IO
' I25 FIGS FIG. 9
Acprzous and organic solution flow Organic soIution flow INVENTORS Thomas D. Kinney John E P. Pickett Porous hydrophilically treated poIyethylene caps ATTORNEY ELECTRON MICROSCOPY TISSUE PROCESSOR CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to co-pending application Microscopy Tissue Receptacle and Method, Ser. No. 658,252, filed Aug. 3, 1967 and which teaches a form of porous tissue receptacle useful in the tissue processing apparatus of the present application. Other applications which are co-pending and which though directed to light microscopy relate in a broad sense to the present invention are Composite l-Iistologic Tissue Receptacle and Embedding Structure and Method, Ser. No. 643,537, filed June 5, 1967, now Pat. No. 3,456,300 and Composite Histologic Tissue Receptacle and Method, Ser. No. 555,640, filed June 6, 1966, now Pat. No.3,430,11l.
BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates broadly to methods, apparatus and devices for processing pieces of tissue but particularly to methods, apparatus and devices related to extremely small pieces of tissue of the size related to electron microscopy practice.
2. Description of the Prior Art The present invention is primarily directed to an apparatus which allows extremely small, i.e. electron microscopy size, tissue particles to be processed while confined in individual porous capsules or receptacles. In comparing the processing of electron microscopy size tissue with the processing of light microscopy size tissue it can be observed that over the years there have been developed a number of highly sophisticated procedures for fixing, dehydrating and penetrating the tissue. Staining for example has been the subject of many special procedures for the purpose of trying to bring out of a particular tissue specimen some desired visually observable characteristic. A comparison of the electron microscopy processing procedures with those procedures employed in processing light microscopy size tissue leads one to the further observation that the specific solutions and procedures employed are dictated primarily by the inherent nature of electron and light microscopy apparatus. For example, processing solutions of a metallic nature might be applicable to light microscopy but not to electron microscopy work.
Conventional methods of transporting light microscopy tissue through the appropriate fixative and processing solutions include placing tissue in tea bags called moss embedding bags or in plastic or stainless steel cassettes and sometimes large pieces are processed in folded gauze. The contained tissues are placed successively by an automated handling apparatus into a series of beakers each containing one of the fixative or processing solutions. The light microscopy processing solutions are of such a nature that they are used in large volume and are reused many times before being disposed of as waste. Upon the completion of this process, the individual tissues are removed and embedded in paraffin, cut on a microtome. stained and viewed under the light microscope. An improved tissue carrier and embedding receptacle which reduces handling of the tissue and also reduces time required for light microscopy work is shown in the referred to copending applications, Ser. No. 643,537 and Ser. No. 555,640.
In contrast to handling comparatively large tissue, i.e.. light microscopy size tissue, the tissue for electron microscopy is normally placed directly into a small, stoppered container whereupon the fixative and processing liquids are added and decanted without removing the tissue though the tissue is necessarily tossed around in the container during the exchanging offluids. At the completion ofthis process the tissue is embedded in an epoxy resin or other medium and later viewed under the electron microscope.
The practice of manually filling and emptying as practiced in electron microscopy tissue processing inherently allows escape of osmium tetroxide fumes with their dangers. The various solution bottles require substantial room for storage and time is lost in pouring from individual bottles into the tissue containers. Care must be taken not to allow absolute alcohol to take up water. Small tissue particles are sometimes lost when solutions are changed. With the present method of exchanging fluids in the tissue container even the moisture in the air can be a problem in that droplets of water may attach themselves to the exposed tissue while fluids are being changed. Overhardening and tissue damage will occur when tissues are left for excessive amounts of time in certain fluids. Fragile tissues may also be damaged by handling of the containers. s. The human potential for error involved in changing fluids at precise times always exists. As the volume of processing tissue for electron microscopy examination has grown the time consuming hand procedure of exchanging processing fluids has not changed, e.g. bottles taken through 10 solutions means 1000 changes. Considerable time is lost when it becomes necessary to change from one solutiontime sequence to another. Every medical laboratory is confronted with large work loads and a shortage of trained technologists. The pressure of insufficient time and diminished supply of competent help must be overcome.
An improvement in the processing of electron microscopy size tissue is provided by use of the porous receptacle disclosed in the referred to co-pending application Ser. No. 658,252. Use of such porous receptacles with manual processing allows a number of minute size tissue particles to be kept separate and separately identified by suitable marking on each respective receptacle while a number of such receptaclesarehnanually processed together in the same container. A saving of the expensive solutions is obtained and exposure to fumes is reduced. Furthermore there is a reduction in tissue damage and the tissue cannot be lost even ifa particular receptacle is accidently dumped out of the processing container since the tissue will beprotec ted by its particular receptacle. Use of such a receptacle with the usual manual method of changing the solutions has shown that with suitable kinds of pores with respect to size and wetting ability, all of the solutions can be exchanged throughthe wallsof the receptacles. With appropriate lag time allowed for penetration and drainage through the walls of the porous receptacles it has also been discovered that only minor modification of the manual processing time schedules is required. That is, in the usual manual processing of electron microscopy size tissue the solutions are poured directly into the container and come into direct contact with the tissue whereas with the porous receptacles the exchange of solutions must be effected through the walls of the receptacles and such exchange introduces some but not a significant time lag. Another advantage that has been discovered through use of the porous receptacles in manual processing is that after the tissue has been run through the usual fixation, dehydration and clearing solutions and is ready for the final Epon, catalyst and like solutions which are used immediately prior to embedding and in which the tissue is frequently held for several hours or even days before final embedding. the tissue may nevertheless be held in the same porous receptacle. That is, it has been found that such a porous receptacle can be used for exchanging all of the usual solutions encountered up to the time the tissue is embedded. Thus, the tissue is not required to be picked up or handled once it has been installed in its particular receptacle until such time as the tissue is to be placed in the final embedding composition, e.g. anepoxy resin.
From the above it can be seen that the use of a porous receptacle for each tissue particle advances the art insofar as manual processing is concerned. Nevertheless, manual processing does not provide a klosed" integral system having all the processing components needed nor does it eliminate the problem of manually exchanging the solutions. The porous receptacles are also still subject to being moved around as the solutions are changed. Sloshing to speed up penetration and tipping of the container to speed up drainage of the receptacles are sometimes required. Furthermore, manual processing does not provide any means for automatically effecting the exchange of solutions, changing solutions, timing solution submersion or for drainning solutions from the receptacles. Used and fresh solutions become mixed.
SUMMARY OF THE INVENTION The invention is directed to an apparatus for processing tissue, particularly electron microscopy sized particles, in which each tissue particle is processed while confined in its own porous receptacle and as part of a closed system and one which provides all of the necessary operating components in an integral unit that can be moved and used together. The porous receptacle may be of a type disclosed in the referred to co-pending application Ser. No. 658,252 and exhibits a porous, wetting, character to both aqueous and non-aqueous, organic, solutions so as to adapt to the various solutions normally encountered in electron microscopy processing preliminary to embedding. A large number of the receptacles may be processed simultaneously in the same processing chamber. The usual manual changing of process solutions is eliminated by providing a series of containers forming part of the processing apparatus and each of which holds a particular solution. Appropriate pipes, electromechanical valves, level controls, and a programming switch are arranged so that predetermined amounts of solutions can be brought to and removed from the processing chamber under any of several solution-time sequence programs. Alternatively, the time sequence may be manually controlled throughout or partially manually controlled and partially automatically controlled. Thus, each tissue particle stays in its particular receptacle throughout the processing as part of a closed system thereby eliminating all risk of damage or loss of the particle when solutions are changed and eliminating exposure of the processing technician to the solutions and sometimes harmful fumes.
Waste solutions are drained to a closed waste tank. To change the solution-time sequence the technician is required only to adjust the programming switch and the number of available programs is limited only by the capacity of the programming switch and the variety of solutions and processing procedures desired.
Penetration of the solutions through the receptacle walls and into the tissue particles and drainage from the receptacles is facilitated by the employment of vacuum in the processing chamber thus eliminating air bubbles and any need to agitate or slosh the solutions or to tip the chamber to facilitate drainage. The processing chamber is shaped so as to be adapted to handling either large or small quantities of the receptacles and which shape also minimizes the amount ofthe sometimes expensive solution which is required in each process step.
The main object of the invention is thus to provide a more efficient and economical way of processing minute size tissue particles.
A more specific object is to eliminate the hand method of exchanging processing solutions when processing minute size tissue particles and the consequent exposure to moisture, tissue damage and hazardous fumes.
Another object is to provide a tissue processor wherein the number and sequence of solutions may be quickly changed and the time of exposure to particular solutions may be accurately controlled either manually or automatically.
Another object is to provide an essentially closed processing system for processing minute sized tissue particles.
Another object is to provide a tissue processing system for small tissue particles which eliminates having to agitate, slosh or tip the particles during processing.
Other objects and advantages of the invention will become apparent when the following detailed description is read in conjunction with the appended drawings and claims. A preferred embodiment of the invention will now be described with reference to the accompanying drawings.
DESCRIPTION OF THE DRAWINGS FIG. 1 is a front elevation view of the tissue processor of the invention.
FIG. 2 is a plan view of the tissue processor of FIG. 1.
FIG. 3 is a fragmentary plan view showing the drain chamber.
FIG. 4 is an exploded perspective view of the capsule chamber and its components.
FIG. 5 is a section view through the capsule chamber showing the relation of the chamber and its components.
FIG. 6 is a fragmentary perspective view of one of the stock fluid containers and solenoid valve control.
FIG. 7 is a fragmentary perspective view of the drain chamber and a portion of the capsule chamber.
FIG. 8 is a perspective view of a porous tissue receptacle suited to use with the invention.
FIG. 9 is a cross-section view of the receptacle of FIG. 8.
FIG. 10 is a schematic diagram ofa control system.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT In reference to the drawings a suitable porous receptacle or capsule 125 for holding the individual particles is generally shown in FIGS. 8 and 9 and can be found described in more detail in the previously referred to co-pending application Ser. No. 658,252. That is, it should be understood that the present invention is directed to a processing apparatus utilizing such a porous receptacle but is not directed to the receptacle per se. In order to provide a porosity suited to the needs of the invention as well as inertness to the solutions, it will be noted that in the form illustrated receptacle 125 has a hollow polyethylene body providing a cavity for the tissue and includes a pair of hydrophilically treated polyethylene end caps 126. 127. Both aqueous and non-aqueous, organic, solution flow may be effected through the end caps whereas non-aqueous solution flow is effected through the body of the receptacle. Unlike bags, gauze pads and the like the somewhat rigid nature of the receptacle prevents collapsing and facilitates both insertion and removal of the particles. As explained in the co-pending applicaiton it is desired that the pores range in average diameter from 40 to 200 microns and the hydrophilic treatment of the end caps. eg by drying ordinary soap, detergent or the like on the caps, insures that all of the solutions normally encountered in electron microscopy tissue processing will find appropriate fluid paths into and out of the receptacle during processing without risking loss of the most minute size tissue particles that are sometimes processed. A typical receptacle of this type might be roughly one-half inch in length and one-half inch in diameter from which it can be seen that substantial numbers of such receptacles may be processed in a relatively small space. It is desirable that individual receptacles be provided in order that they may be handled individually during embedding. However, while not shown it is contemplated that several such receptacles may be molded and used as an integral unit.
As previously mentioned the tissue processor of the invention is basically directed to a closed system which allows a plurality of receptacles 125, each containing one or more minute tissue particles to be processed, according to a scheduled time sequence, through various'selected processing solutions. The particular solutions and the particular time sequence may vary according to different programs.
Referring to the drawings and particularly to FIGS. 1, 2, and 3, tissue processor includes a cabinet 11, a capsule processing chamber 12, stock solution containers 13, I4, l5, 16, I7, 18, 19, 20, 21, and 22 and a closed drainage container 23. For purpose of the signal lights later referred to the containers carry operational numbers 1 through 10.
Processing chamber 12 remains stationary and is adapted to receive a plurality of the porous receptacles 125. As best illustrated in FIGS. 4 and 5, chamber 12 includes a container 25 having a funnel shaped bottom which slopes towards a central drain opening. This sloping shape facilitates rapid draining and when only a small number of receptacles are to be processed reduces the amount of fluid required to submerge such receptacles. For example, as shown in FIG. 5 the receptacles indicated in solid lines might represent such a small number of receptacles requiring only a low level of processing solution whereas with a larger receptacle load as indicated by the receptacles shown in dashed lines a higher level of processing solution may be employed. A suitable perforate disc 48 provides weight sufficient to overcome the bouyancy of the receptacles and keeps them submerged.
The stock solution containers are each connected to the chamber 12 by individual conduits, flow through which is controlled by electrically operated valves. Pneumatic, mechanical or other valve devices may be employed for the same purpose. A typical container, conduit, valve arrangement is illustrated in FIG. 6 where container 22 (having an operating number 10) is shown having a quick disconnect 74 to facilitate changing containers and a tube 75 connected to chamber 12 and controlled by valve 76. The respective conduits communicate with holes 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 around the upper part of container 25. A separate hole 36 is adapted for attachment to a suitable vacuum source 131 such that processing in chamber 12 may be effected under vacuum. Use of vacuum has been found to speed up penetration through the receptacles, to eliminate bubbles in the receptacles and to avoid the need for mechanical sloshing or agitation. Vacuum source 131 preferably forms part of an integral processing system so that processor 110 may be readily moved and operated as a unit. A separate vacuum source may of course be employed.
To facilitate removal of a group of the processed receptacles a perforated basket 40 conforms to the internal shape of container and may be removed at the conclusion of any selected processing program. It will be noted that basket 40 is provided with holes 26a, 27a, 28a, 29a, 30a, 31a, 32a, 33a, 34a, 35a, and 36a all of which correspond with the respective holes 26 through 36 in container 25. A slot 41 is formed in basket and aligns with a pin 42 on the inner wall of container 25. Slot 4] and pin 42 thus locate basket 40 so that the various holes 26 through 36 and 26a through 36a are properly aligned.
Processor 110 is adapted to be operated so that all solution admission, solution retention and solution drainage takes place in automatic timed sequence. Alternatively, processor 110 may be operated so that each solution admission, retention and drainage may be manually controlled both with respect to sequence as well as time. When on automatic operation the processor provides a choice between high and low levels in chamber 12 and is thus adapted to handle either relatively large or relatively small quantities of receptacles and with a minimum requirement for the respective processing solutionsr One means for sensing level and shutting off solution flow is provided by the indicated probe sensor rods 43, 44 and grounding rod 45 and which may be connected to a suitable electronic liquid level control 130 such as the sensing and amplifying circuit made and sold by Cole-Parmer Instrument & Equipment Company, 7330 North Clark Street, Chicago, lllinois 60626. The level sensing can be switched to either high or low level by a level selector switch 107 and in the case of high level sensing, achieving ofthe correct level depends upon the establishment of an electrical path between grounding rod 45 and sensor rod 43. Where low level sensing is being employed sensing depends on the achieving of an electrical path between grounding rod 45 and sensor rod 44.
During processing an essentially airtight cover for chamber 12 is provided by a preferably weighted top 47 which rests on an O-ring 46 embedded in the top of container 25. Top 47 includes a transparent disc section 47' which allows the technician to visually observe the interior of chamber 12 and the receptacles being processed.
Drainage of chamber 12 is controlled by a solenoid valve 61 which allows the used solutions to be dumped into a closed waste container 23 for holding pending ultimate disposal. Container 23 is located within cabinet 11 and has a pair of pipe connectors 62, 63 which receive respectively a drain line coming from chamber 12 and a vacuum line 81 connected to the vacuum source 131 and which speeds up drainage from chamber 12. In order to prevent overflow of container 23 a level sensing system similar to that previously described may utilize a sensor rod 82, a grounding rod 83 and a level control 132 with provision for connection to a suitable warning light 133, buzzer or the like. A suitable drain spout 24 allows container 23 to be periodically emptied of the various accumulated used solutions.
The previously referred to solution containers 13 through 22 (with corresponding operating numbers 1 through 10) have respective caps 50, 51, 52, 53,54, 55, 56, 57, 58, and 59 for refilling the respective containers. Suitable air vents 60, 61, 62, 63, 64, 65 and 66 are provided but are preferably kept extremely small so as to limit any admission of moisture. Some solutions such as absolute alcohol, propylene oxide and propylene oxide plastic require special care and are kept substantially moisture free by means of special cap containers 67, 68 and 69 which are adapted to hold a desiccant material and are further provided with extremely small vent holes 70, 71 and 72. Compared with the conventional method of hand pouring each solution as it is required, it can be seen the the inventions method of storing and handling solutions offers many operating advantages. Furthermore the illustrated rectangular shaped solution containers as compared to the usual bottle containers provides a means for storing all of the solution in a relatively small space and enabling the processor to be manufactured and used as an integral unit. Only fresh solution is stored and only fresh solution passes through the container to chamber lines.
The front of cabinet 11 effectively acts as a control panel and mounts the various signal lights, control switches and the like. An on-off switch 89 controls power to the processor and when on energizes the vacuum source pump B1 and a suitable signal light 90 to indicate to the operator that the processor is energized. Selective manual or automatic operation is provided by the selector switch 91. Manual control of each of the respective container solenoid flow control valves is achieved by the manual on-off switches 92, 93, 94, 95, 96, 97, 98, 99, and 101 and which control flow from containers l3, l4, l5, l6, l7, 18, 19, 20, 21 and 22, respectively. Corresponding signal lights 92a, 93a, 94a, 95a, 96a, 97a, 98a, 99a, [00a and 101a are energized on both manual and automatic operation whenever a solution is being drawn. The signal lights, manual switches and program switch 106 are arranged so that the signal light which is on shows either the solution being drawn at the time or the solution which is in chamber 12. Signal light changes are coordinated with drainage. Drainage of chamber 12 is manually controlled by a panel switch 105 which controls solenoid valve 61 or is automatically controlled by switch 106.
It has been mentioned that the art of processing minute size tissue particles for electron microscopy examination has led to a great variety of programs or time sequence-solution procedures. Medical authorities differ in the specific solutions and time sequences which they regard as being best suited to particular tissue conditions. Therefore any processing apparatus such as provided by the invention must be adaptable to any of a variety of solutions and to any of a variety of time sequences. The present invention is based on a sequence requiring not more than ten solutions and for automatic operation is based on the possibility of three separate programs. The number of solutions may of course be increased simply by increasing the number of stock solution containers, valve controls and the like and the number of program possibilities may be increased simply by expanding the switching time capability. it is also contemplated that a number of separately controlled processing chambers may be employed as part of the same overall system.
As examples of three typical programs the following three programs are listed:
1. Glutaraldehyde, 4%
minutes. 2. Rinse in butter 12 minutes.
Program I Program 11..
ln a typical manual operation, the various valve control switches are turned on and off in the desired sequence and level and drainage are controlled by visual observation. Time is controlled by reference to a clock or the like and vacuum is employed, When on automatic operation, the valve control switches are controlled by a suitable programming switch 106 such as that sold by the Sealectro Corporation, 225 Hoyt Street, Mamaroneck, New York. As previously referred to, level in the chamber 12 may be controlled by the respective sensor rods 43, 44 and choice of high or low level by the level selector switch 107.
Under automatic operation the programming switch 106 is set to the desired program I, ll, or III upon energization of the processor circuitry vacuum will be applied and the particular solenoid valve controlling the first solution to be introduced will be energized by switch 106. However, once the desired level in chamber 12 has been achieved, depending on the setting of the level selector switch 107. the particular selected solenoid valve will be deenergized so as to shut off the first selected solution. The program switch 106 will of course be rotating at some predetermined rate and at some predetermined time will energize solenoid valve 61 such that chamber 12 is drained. Following a predetermined time allowed for such drainage, solenoid valve 61 is deenergized and the next respective container solenoid control valve is energized so as to admit the next required solution into processing chamber 12. The same control sequence will follow; that is, the particular solenoid valve will be energized and fluid will be admitted to chamber 12 until the selected high or low level is achieved whereupon the level control will cause such solenoid valve to be deenergized and the respective solution flow shut off.
In some applications it is desirable to hold the tissue in the receptacles in the first solution for several hours prior to initiating a particular time-solution program sequence. For such purpose a long time delay switch 108 may be employed so that the first solution may be admitted manually and the remainder of the particular program on an automatic basis after some predetermined time delay, such as in the order of four hours.
While a wide choice of materials are available with which to construct the various components of the apparatus of the invention it will of course be apparent that some of the solutions required are ofa toxic nature and furthermore are ofa corrosive nature with respect to certain materials. The choice of material employed in constructing the container, conduits, solenoid valves, processing chamber and the like should keep these factors in mind. It is further contemplated that other level sensing means might be employed based on sensing the position of a magnetized float within chamber 12. In this instance the chamber itself should preferably be formed of some material that would be of a non-magnetic nature. It is also contemplated that photocell detectors may be employed for level sensing.
While the invention is primarily intended to provide means for processing minute size particles for subsequent electron microscopy examination it will be apparent that the processor of the invention may be used for processing the larger size tissue particles which are intended for light microscopy examination. In either case the required aqueous and non-aqueous, organic, fixation, dehydration and clearing solutions may be installed in the respective solution containers and introduced as reqiuired for the particular type of microscopy examination. In this regard, it should be noted that the practice in electron microscopy work is not to reuse the solutions and in the system of the invention only fresh solution is transferred through the lines and valves connecting the containers with the processing chamber. If the particular solutions are required to be pumped back to the containers after use appropriate pumping and switching controls would have to be provided.
With further regard to the choice of solutions for a particular processing sequence it should also be noted that the described form of sensing level depends on the ability of the solution to electrically conduct. Propylene oxide, absolute alcohol and percent alcohol, in particular, give a more dependable level sensing response when made up with 1 /2 percent, by volume, stock Epon. Other solutions may require similar dilution to enhance the conductance. Of course, where photo-electric or magnetic level sensing is employed the conductance as such need not be a consideration.
While a particular embodiment of the invention has been described it will be appreciated that the above mentioned alternate and other embodiments will become apparent to those skilled in the art and yet be within the spirit and scope of the invention as hereafter set forth in the claims.
1. A closed processing system enabling a plurality of individual particles of minute electron microscopy size, having at least one dimension no greater than one millimeter, to be separately contained while being bathed simultaneously for varying lengths of time in successive aqueous and non-aqueous electron microscopy tissue solutions so as to fix, dehydrate and clear the particles preparatory to embedding, comprising:
a. a plurality of uniform porous tissue receptacles each being adapted to contain and physically isolate a group of said particles having at least one particle per group, each receptacle having a porous body portion defining an open cavity adapted to receive and retain a group of particles and porous closure means removablyreceived by said body to enclose said cavity, the material forming said receptacle having pores of average diameter from 40 to 200 microns throughout said receptacle, being buoyant in and inert to all said solutions and sufficiently rigid to prevent collapsing during processing, being inert to all of said solutions and having for each of said solutions at least some portion permeable thereto;
b. a plurality of closed stationary solution containers each containing a particular electron microscopy tissue processing solution and collectively containing all of said aqueous and non-aqueous solutions;
c. a stationary discharge pipe and associated valve means.
for each container connected to provide an individual selectively controllable flow path for each of said solutrons;
d. a stationary processing chamber mounted adjacent said containers, said chamber having a removable top cover and being adapted to receive and process selected small and large numbers of said receptacles simultaneously, said processing chamber being operatively connected to each said container pipe and valve means such that admission of each said solution to said processing chamber may be independently controlled by opening and closing the appropriate said container valve means and such that said solutions may reach said chamber with substantially no mixing therebetween;
e. submersion means adapted to fit within said chamber and adapted to the selected number and buoyancy of said receptacles for maintaining said receptacles substantially stationary and successively submerged in each respective said solution;
f. drain pipe and valve means operatively connected to said chamber and enabling said chamber to be drained upon the opening thereof; and
g. control means for remotely operating said container and drain valve means in some predetermined time sequence whereby with a selected number of said receptacles installed in said chamber and said cover on said chamber said solutions may be successively and independently admitted to said chamber in some predetermined volume, retained for predetermined times and drained from said chamber in a corresponding processing sequence such that each of said aqueous and non-aqueous solutions is exchanged with each said group of particles by penetration and drainage through said pores while maintaining said system closed, and each said group substantially stationary and physically isolated.
2. A system as claimed in claim 1 in which said containers, pipes and chamber are unheated and adapted to said solutions being maintained and said processing sequence in said chamber being conducted at ambient temperature;
3. A system as claimed in claim 1 wherein said processing chamber provides an inwardly tapered bottom adapted in size and shape to hold a predetermined small said number of said receptacles fully submerged with a minimum requirement of said solution volume and said submersion means adapts to said bottom and number.
4. A system as claimed in claim 1 including a vacuum source operatively connected to said processing chamber and producing a vacuum effect operable through said receptacle pores to accelerate penetration of said receptacles and the respective groups of particles therein by said solutions, and means to hold said vacuum throughout said processing sequence but at a value insufficient to collapse said receptacles.
5. A system as claimed in claim 1 adapted to each said solution being retained substantially static and unagitated during said sequence.