US3771490A

US3771490A - Automatic tissue processor

Info

Publication number: US3771490A
Application number: US00228115A
Authority: US
Inventors: T Kinney; J Pickett
Original assignee: Individual
Current assignee: Individual
Priority date: 1972-02-22
Filing date: 1972-02-22
Publication date: 1973-11-13
Anticipated expiration: 1990-11-13

Abstract

A tissue processor is programmed for fixation, dehydration and clearing of electron microscopy size particles and utilizes a porous receptacle for each particle or group of particles of tissue and a processing chamber adapted to contain a plurality of the receptacles. The chamber is connected to a plurality of containers, some of which are refrigerated, and which contain the various processing solutions. The solutions are individually piped to, and from beneath, the chamber through a remotely controlled valve and manifold arrangement which also connects with a metering pump. This arrangement minimizes fluid contamination and allows each solution to be precisely metered, brought to, retained in and drained from the chamber thus allowing the particles to be bathed in the solutions according to an automatic programmed time sequence. The program may be varied as to number of cycles per solution, as to number of solutions per program, as to time per cycle and as to starting and terminal solutions in the program.

Description

United States Patent Kinney et al.

[ AUTOMATIC TISSUE PROCESSOR [76] lnventors: Thomas D. Kinney, 3120 Devon Rd.; John E. P. Pickett, 3323 Pinafore Dr., both of Durham, NC.

22 Filed: Feb. 22, 1972 [21] Appl. N0.: 228,115

[52] U.S. Cl ..1l8/7,118/421, 222/136 [51] Int. Cl. B05c 11/10 [58] Field of Search 118/4, 7, 8, 421, 118/429, 50; 134/96, 97, 98, 195; 137/1 NE; 222/136, 144.5, 144, 190; 95/93 [56] References Cited UNITED STATES PATENTS 2,774,364 12/1956 Brobeil 222/136 X 2,959,151 1l/1960 Ehrlich 118/429 3,092,121 6/1963 Broge 134/97 X 3,227,130 1/1966 Weiskopf 118/11 3,294,101 12/1966 Suzuki et al.. 134/95 X 3,392,780 6/1968 Brown 118/429 3,400,726 9/1968 DuGrail 134/95 3,452,710 7/1969 l-lentschel 118/7 3,526,203 9/1970 Kinney 118/7 3,550,552 12/1970 Archibald 1123/? O WASTE Primary ExaminerMorris Kaplan AttorneyB. B. Olive [57] ABSTRACT A tissue processor is programmed for fixation, dehydration and clearing of electron microscopy size particles and utilizes a porous receptacle for each particle or group of particles of tissue and a processing chamber adapted to contain a plurality of the receptacles. The chamber is connected to a plurality of containers, some of which are refrigerated, and which contain the various processing solutions. The solutions are individually piped to, and from beneath, the chamber through a remotely controlled valve and manifold arrangement which also connects with a metering pump. This arrangement minimizes fluid contamination and allows each solution to be precisely metered, brought to, retained in and drained from the chamber thus allowing the particles to be bathed in the solutions according to an automatic programmed time sequence. The program may be varied as to number of cycles per solution, as to number of solutions per program, as to time per cycle and as to starting and terminal solutions in the program.

18 Claims, 11 Drawing Figures PATENTEmmv I 3 I975 3771,2190

SHEET 2 OF 5 o 11K g I; I \1y 3- 7P, 1, H 11-; "I I}; II. CYCLES nufluullfluuil 17O I? I a 1 I HOLD/PASS ililuflflfllsfllifl f) I I TIME nmaan smrm m m E m r 5 160 I 155 I40 4 7 SOLUTION INDICATOR "HMER I 2 4 5 e 7 8 9 10 a H I 3 8 DIIIIIIIIIEIIIIEIDEII] '4 2 9 132, I ISO I; ON

I 10 :na 5 C RELEASE 11 I OFF SELECTOR A o D 7s 9 1530 VOLUME I 181 IIIIIIIIIIIII 120 (a 98/0 95/0 96/6 979 II I l I I 82 I A a FIG. 5

I72) I I I I! v E 1 46V m m1 SOL I :QOL. 1 2 gQ 4 3 41, 3.2T 0604 40% a LT, H ALC. I I f 2 9y/ I [.1-'-'JYz- .1\l v I. .'v. ;l-. I I I OQEQ-NC SOLU'ION LOVV 2 ORGANIC SOLUTION FLOW SPECIMEN NUMBER PAIENIEMM 13 ms SHEET 3 BF 5 jDu mkm OP PAIENTEnunv 131915 3.771. 490 SHEET u 0F 5 E IG. 8

TO WASTE AUTOMATIC TISSUE PROCESSOR CROSS REFERENCE TO RELATED APPLICATIONS This application is related to copending applications Ser. No. 860,256, filed Sept. 23, 1969, now U.S. Pat. No. 3,697,299 entitled Microscopy Tissue Receptacle Method and Ser. No. 60,798, filed Aug. 4, 1970, now abandoned entitled Electron Microscopy Tissue Processing Method.

BACKGROUND or THE-INVENTION 1. Field of the Invention This invention relates broadly to apparatus and devices for processing pieces of tissue but particularly to apparatus for processing electron microscopy size tissue.

2. Description of the Prior Art This invention constitutes an improvement upon the subject matter of our prior U.S. Pat. No. 3,526,203, upon the subject copending applications and upon the methods related thereto. Since the general history of the prior art is dealt with in some detail in prior U.S. Pat. No. 3,526,203 reference is made to U.S. Pat. No. 3,526,203 for the prior art background of the present invention. Reference is also made to prior U.S. Pat. No. 3,536,040 directed to a selectively permeable receptacle and to U.S. Pat. No. 3,576,176 directed to a tissue capsule rack which forms part of the prior art of specific interest and useful in practicing the present invention. In summary, the subject U.S. Pat. No. 3,526,203 discloses a processor for processing electron microscopy size tissue, as well as larger tissue, in a permeable receptacle of the type disclosed in U.S. Pat. No. 3,536,040 with the receptacle being held in a rack as shown in U.S. Pat. No. 3,576,176. Our own prior art is believed to be the closest known prior art. Of particular interest to the present invention is that the processor of our prior patent disclosed a system of connecting the processing chamber with the processing solution containers by means of pipes which connected to the top of the processing chamber and depended on gravity feed and further depended on sensing the level of the solution in the chamber as a means of stopping flow and measuring the volume of a particular solution. After extensive experimental use it was found that the processor of our prior patent inherently introduced substantial opportunity for the solutions to mix in the various pipes between the chamber and the containers. Also, after experimental use with various types of solutions, particularly osmic acid, and time programs the prior processor was found to introduce a problem in maintaining accurate level control for the various solutions. Certain solutions when passed through relatively long lines tended to precipitate in the lines particularly when the solution was held at or=above room temperature and no provision was made in the prior processor for maintaining selected solutions at temperatures below room temperature.

Of particular interest is the fact that while our prior patent taught the general concept of automatic operation with a program switch the processor of the prior patent did not disclose means for conveniently changing the numbers of cycles for a particular solution. That is, in hand processing it is sometimes the practice to hold the tissue in a solution, then drain the solution, then hold the tissue again in the same solution for another time period. The processor of our prior patent did not provide a means for duplicating in a machine operation this aspect of hand processing. While the prior patent processor contemplated changing an entire program in order to obtain such a variation in numbers of cycles per solution the processor of our prior patent did not not offer the possiblity of making such a change even while a particular program was in progress. Furthermore, the program capability of the processor of our prior patent was to some extent limited in being able to vary the amount of time devoted to a particular solution being in the processing chamber. Thus, with the processor and processing method of our prior patent if it was desired to change the base time during which a particular solution was held in the processing chamber from, say, 12 minutes to 24 minutes it was necessary to change the entire program for the entire run of solutions. Also, the prior processor did not exhibit the capability of being able to pass certain solutions in a program or to terminate a particular program, at least temporarily, with a particular solution. In hand processing of electron microscopy tissue it is sometimes desirable to, say, stain a tissue after processing

solutions

1, 2 and 3 and to, say, bypass solution 4 with a certain tissue.

From the foregoing it can thus be seen that while the processor of our prior patent constituted an important advance in the art and so far as we know constituted the first significant step toward a practical automatic processor for processing electron microscopy size tissue long and extensive experimental use with that processor have revealed a number of needed improvements in terms of both apparatus and such constitutes the object of the present invention.

SUMMARY OF THE INVENTION The invention is generally directed to an apparatus for processing electron microscopy size tissue and constitutes an improvement over the apparatus shown called for in our prior U.S. Pat. No. 3,536,203. Reference to our prior patent is made for an understanding of the general operation involved with an apparatus of this kind. Of specific interest to the present invention, the apparatus embodies a stationary processing chamber having a bottom port connected directly to a manifold which is positioned below the chamber. The manifold is also provided with a plurality of inlets connected through pipes to each of the respective processing solutions and with an outlet for waste discharge. Each inlet on the manifold mounts a remotely solenoid controlled valve so that the solutions from each solution container can be effectively isolated from each other at points immediately adjacent the manifold which minimizes the opportunity for, say, solution 1 to mix with solution 3 and also minimizes the amount of necessary waste solution. The chamber port communicates with a solenoid control valve immediately adjacent the manifold as does the waste outlet. A metering pump also connects to the manifold and incorporates a metering piston driven by a reversible motor so that the piston can be withdrawn to pull in a given amount of solution and can be moved in the opposite direction to force the solution into the processing chamber through the mentioned bottom port. The manifold thus provides a passage for a selected solution to be withdrawn into the pump and to then be forced into the processing chamber. During withdrawal all valves except the selected solution valve are closed. On the return stroke only the processing chamber valve is opened and during dumping only the waste valve is opened to allow the expended solution to drain by gravity. Means are provided whereby the amount of pump cylinder movement can be precisely regulated to vary the amount of solution withdrawn so as to eliminate the need for level sensing and control as in the processor of our prior patent. The various solenoid valves are also coordinated by program switch means such that all the respective solutions may be metered, pumped up to the processing chamber and then allowed to drain by gravity to waste with minimal mixing of one solution with another except in the collective waste.

For greater variety of programming, the invention also adopts the concept of establishing a so-called base time of, say, 15 minutes and incorporating means whereby a particular solution may be held during a particular cycle for one, two or three periods of base time. Thus, merely by moving a switching arrangement asolution can be held for, say, 15, 30 or 45 minutes. This can be further varied so that, during a particular program, solution 1 stays for, say, 15 minutes, solution 2 for 45 minutes and solution 3 for 45 minutes in that sequence. The program can be changed while in progress or from program to program merely by reorienting a set of switching positions. Thus, a medical research investigator is offered a wide range of processing techniques for comparing one mode of processing the same tissue or different tissue particules with another mode of processing. The processor of the invention also mounts a switching arrangement adapted to allow any particular solution to be bypassed in a program or for the processing to stop, at least temporarily, when such solution is reached. An improved ventilation system has also been provided which allows -fumes in the work area and particularly around the processing chamber to be withdrawn as rapidly as formed.

In summarizing the present invention in apparatus terms, it can be said that the programming capability has been substantially increased, the opportunity for solution mixing has been minimized and control over the quantity of solution used has been made much more precise. In method terms, it can also be said that the method of the invention minimizes intermixing of solutions, increases accuracy of measuring and provides greater variety in programming. Other improvements of an important but lesser nature will also be noted as the description proceeds.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of an apparatus for processing electron microscopy tissue according to the invention.

FIG. 2 is a right elevation view of the apparatus.

FIG. 3 is a rear elevation view of the apparatus.

FIG. 4 is a left elevation view of the apparatus with various compartment doors open.

FIG. 5 is an enlarged front elevation view of the main control panel and also showing the auxiliary control panel in an open position.

FIG. 6 is a front elevation view of the solution containers with the container compartment doors being shown in an open position.

FIG. 7 is a perspective somewhat schematic view of the manifold, metering pump, processing chamber and waste tank arrangement.

FIG. 8 is a sectional and somewhat schematic elevation view of the chamber, valveand metering pump arrangement.

FIG. 9 is a plan, sectional and somewhat schematic view of the manifold, passages, valves and metering pump.

FIG. 10 is a schematic diagram of the control system.

FIG. 11 is a perspective view of a prior art porous receptacle used with the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention is directed to processing apparatus and a method for processing tissue particles held in a porous receptacle 25 of the type shown in FIG. 11 and more fully disclosed in related US. Pat. No. 3,536,040. A typical receptacle of this type is porous at least in some portion, whether in the body 20 or the end caps 21, to all the solutions. It may be roughly onehalf inch in length and one-half inch in diameter and the pore size in the range of about 40 to 200 microns. A substantial number of such receptacles may be processed simultaneously and according to a time schedule for the various processing solutions as later discussed.

The processor of the invention includes a cabinet 29 having pivoted

front doors

30, 31, a pivoted rear door 32 and a pivoted top door 33.

Doors

30, 31 cover, respectively, a refrigerated compartment 40 and a nonrefrigerated compartment 41 having a dividing partition 42 and a back wall partition 43. Suitable heat insulation 45 is mounted on door 31 and surrounding compartment 40 in order to retain the reduced temperature obtained by suitable refrigeration mechanism generally indicated at 46. Ten solution containers 50-59 are shown by way of example and are labeled respectively:

solution

1, 4 percent glutaraldehyde; solution 2, buffer;

solution

3, 1 percent osmium tetroxide;

solution

4, 50 percent alcohol; solution 5, percent alcohol;

solution

6, 80 percent alcohol;

solution

7, 95 percent alcohol; solution 8, absolute alcohol; solution 9, propylene oxide; solution 10, propylene oxide-Epon 50-50. The first three mentioned solutions have been found to require refrigeration for best processing results and it is for this reason that compartment 40 is refrigerated. Each solution container is provided with a suitable quick-disconnect 70, FIG. 6, and an air inlet 71 to allow the solution to be withdrawn through a connecting tube 72. The respective connecting tubes 72 are each connected to a respective solenoid valve 73 which in turn are mounted on a manifold structure 75. Structure 75, as shown in FIG. 9, provides a series of short passages for communicating all of the various solution, chamber, waste and pump inlets and ports. Like other components exposed to the solutions, structure 75 must be of a material inert to all the solutions and stainless steel and Teflon plastic have been employed. The ten tubes 72 leading from the ten solution containers 50-59 are connected to ten respective solenoid valves 73. Manifold 75 also mounts above the manifold a solenoid valve having a connecting pipe 81 connected to the processing chamber 90 and a pipe 82 connected to the manifold 75. Below the manifold there is mounted a further solenoid valve 85 having a pipe 86 connecting to the manifold 75 and a pipe 87 connecting to a waste tank 88. The exposed portions of the valves must be inert and are preferably stainless steel with Teflon inserts though other materials may be employed if suitably inert to the solutions.

Manifold is also connected through a pipe with a pump metering chamber generally indicated at 101. All parts of the metering pump exposed to the solution should be relatively inert. Teflon plastic and stainless steel have been employed for this purpose. Chamber 101 is formed in a piston operated metering pump which is designated 99 and in this case includes a reversible electric motor 105, FIG. 7, connected to drive an internally threaded tube member 106 which in turn receives a threaded piston rod 107 such that when the armature of motor rotates in one direction piston rod 107 is moved forwardly to move piston 110 toward the cylinder port 111 and when the motor armature turns in the opposite direction piston 110 is moved away from port 111. Piston rod 107 mounts a pin 112 which moves back and forth in a slot 113 located in bracket 114. Four micro-switches 115, 116, 117 and 118 are mounted in the path of retracting movement of pin 112 so as to strike pin 112 and be actuated. Switches 115, 116, 117 and 118 are arranged in the circuitry so that one of the switches can be selected to control the amount of movement of pin 112 during retraction by deenergizing motor 105 and by this means very precisely control the amount of solution metered by pump 99 and indicated by levels A, B, C and D. It should be understood that appropriate circuitry commands motor 105 to turn to advance piston 110 when a solution is to be metered, other circuitry commands piston 110 to retract, other circuitry selectively controlled by micro-switches 116-118 command motor 105 to stop and other circuitry commands motor 105 to turn so as to advance piston 110 to pump out the metered solution. Annular piston edges 109 are formed to assist sealing.

The processing chamber 90 of the present invention is generally like but differs in an important respect from that shown in prior US. Pat. Nos. 3,526,203 and 3,576,l76. Specifically, the solutions, instead of being admitted at the top through plural inlets, are both admitted and discharged through a single port 91 in the bottom of chamber 90. Level sensing is thus avoided by means of the described metering pump. Chamber 90 has a tapered bottom which assists in dumping solutions and also allows a relatively small number of capsules to be processed. To describe the processing chamber 90 in more detail it will be seen that the removable cap 120 is provided with a clear glass window 121. Chamber 90 is physically located within cabinet 29 near refrigerated compartment 40 which minimizes solution precipitation, particularly that of osmium tetroxide. Cap 120 is positioned slightly above the horizontal cabinet wall 123 which provides a relatively large unobstructed work surface to the side of processing chamber 90. The chamber should be inert to all solutions and has been made of Teflon plastic. Stainless steel and other materials may be used. As shown in FIG. 8, a small vent hole 124 is provided on the upper inner wall of the processing chamber which allows the solutions to drain and also allows the fumes from solutions to be exhausted through an outside hole 125 by an exhaust fam connected through a flexible conduit 131 to a suitable hood, not shown. That is, any fumes escaping through

holes

124, 125 are drawn into the interior of cabinet 29 and then exhausted by fan 130. Also, when cap 120 is removed any fumes escaping from chamber 90 are drawn into apertures 126 in top wall 123 as shown in dotted lines in FIG. 1. The fumes are then drawn inside cabinet 29 and exhausted by fan 130. During processing the capsules are secured in a tissue capsule rack of the type described in US. Pat. No. 3,576,176, indicated at 92, FIG. 8.

The processing of tissue particles according to the invention has been described as generally involving placing the tissue particles in the receptacles; placing the receptacles in the receptacle rack, placing the rack in the chamber and then drawing the solutions into the processing chamber in some predetermined time sequence. In this regard the present invention elminates level sensing in chamber 90 and allows the amount of each fluid drawn into the processing chamber to be precisely metered according to the number of receptacles being processed. Thus, the panel board in FIGS. 5 and 10 will be seen to have a volume control switch 132 marked A, B, C and D which correspond in one embodiment to respective loads of one receptacle, (lOmL), two to four receptacles (25 ml.), five to 28 receptacles (50ml.) and 29 to 48 receptacles (100 ml.). Switch positions A, B, C and D effectively select a corresponding micro-switch from the group of pump microswitches 115, 116, 117 and 118 and thereby control the level of fluid used of a particular solution. Additional program circuitry, not shown, commands the pump to otherwise start and stop as required.

The circuitry also includes what is called a cycle control which is shown at 135 in FIGS. 5 and 10 and which allows either 1, 2 or 3 solution changes of each stock solution. That is, the group of ten, three position switches shown in FIG. 5 and 10 at 135 are suitably connected into the illustrated program control circuitry so that each solution can be controlled during a particular program as to the number of changes I, 2 or 3 of that solution. Thus, the cycle switch for solution 1 may be set to call for solution I to have one change, the cycle switch for solution 2 may be set to call for solution 2 to have two changes, the cycle switch for solution 3 may be set to call for solution 3 to have one change and so forth, as shown in FIG. 10. Furthermore, cycle switches may be set one way for one program and immediately after that program is terminated the switches may be moved to a different array for the next program. In this manner, a versatility not heretofore achieved is provided. Those skilled in the art will, of course, understand that the cycle switches by their settings control whatever program circuitry is employed. FIG. 10 is intended to simply illustrate one such configuration based on using a stepping switch and standard logic type circuits.

In another aspect of the programming, the present inention also provides for a so-called time control." The time control operates on the basis that each program is made up of some multiple of a standard base time, e.g., 15 minutes. A switch 150, shown in FIGS. 5 and 10, is a multiple position timing switch and each position corresponds to a portion of the base time period of 15 minutes. That is, switch can beset for any time from zero to 15 minutes. In addition to switch 150 the time control also includes for each of the 10 solutions a multi-position time switch indicated at 155, which can be moved to a position to indicate either one, two, three, four, five, six etc., base units of time. Thus, if the timing switch 150 is set on ten minutes and the particular time switch for solution 1 is set on the 2 position this would mean that solution 1 would stay in processing chamber 90 for 20 minutes (two times the base 10 unit) and then run out. That is, each cycle with solution 1 would be for 20 minutes. If the corresponding cycle control switch 135 for solution 1 were set on its 1 position the exchange of the solution I with the particles would only take place once but if, for example, the cycle control switch 135 were set on, say, its 2 position the exchange would be repeated for another twenty minute period. The base time control circuitry as well as the cycle control circuitry is, of course, connected to suitable programming circuitry as generally illustrated in FIG. 10. Thus, any solution can be regulated independently of all other solutions as to the number of cycles and the time per cycle merely by positioning the

switches

135, 150 and 155.

The circuitry of the invention also incorporates a set of ten, three position, hold-pass switches 170 having positions indicated by N for normal, P for pass and S for stop. Switches 170 are suitably connected into the program circuitry as indicated in FIG. 10. As background information it may be noted that tissue processing of electron microscopy size tissue is subject at times to many collateral operations for experimental purposes or to get certain results supplementary to those achieved by routine processing. That is, insome cases a program can be set up which simply calls for the tissue particles to be processed step by step through all the solutions. In other cases however the research investigator may, for example, want to bypass certain solutions or he may want to, at least temporarily, terminate the program after a particular solution. When the particular hold-pass switch 170 is set on N for normal the solution is used in a normal way and the switch position has no effect on the program. However, when a selected hold-pass switch 170 is set on P this switch setting directs the program circuitry to pass the respective solution in the program and not use it. Thus, if the hold-pass switch 170 is set on P for solution 3, this solution would not be used at all. Also,if the particular hold-pass switch 170 is set on S the associated program circuitry is so arranged as to call for the program to shut down immediately after such solution has been dumped. Thus, if the operator technician is directed to stain all the particles in a special stain solution between, say,

solutions

6 and 7 the technician may set the hold-pass switch 170 corresponding to solution 6 to the S position and the program will temporarily stop after solution 6 has been dumped to allow this collateral staining operation. g I

Considering further the circuitry and control panel shown in FIGS. and 10, there is also incorporated a selector switch 160 having ten positions, corresponding to the ten solutions, and which allows the operator of the processor to select as a starting point in any program any of the ten respective solutions. Thus, a particular tissue may require special processing, in only, say,

solutions

7, 8, 9 and 10 and this can be immediately ob- Y tained dependent only on a proper initial setting switch 160.

To complete the general explanation of the circuit it will be understood that given the concept of providing switch means to vary the base time, the cycles per solution, the particular solution starting point, the time per cycle per solution and processing chamber level that many and varied logic, stepping switches and other program and timing arrangements other than that shown generally and substantially in FIG. 10 may be employed in conjunction with such switch means. That is, the 10 cycle switches 135, the 10 hold-pass switches 170, the 10 time switches 155, the selector switch 160, the volume switch 132 and the base time switch 150 may be used to control a variety of program circuits suitable to the invention and well known in the art of programming. The circuitry also includes a start switch 180 whose purpose, and circuit arrangement, is to start a selected program once the various program selected switches have been positioned as required for such program. In addition there is provided a release switch 181 and a so-called clean switch 182. The purpose of release switch 181 is to provide a means to operate the dump valve so that in the event of a mulfunction or for other reasons it becomes necessary to immediately drain chamber this can be done by actuating release switch 181. Switch 181 is wired accordingly to override all other program commands. The clean switch 182 serves another useful purpose and is wired in the circuitry so as to command only certain solution control valves 73 to operate in some predetermined sequence designed to flush or clean out the chamber 90, manifold 75 and the various passages at the end of one program and before another program starts. Thus, clean switch 182 effectively commands a subsidiary program intended for cleaning the system and not for processing as such. In one embodiment clean switch 182 and the associated circuitry are programmed to dump fluid from chamber 90, then to rinse twice with propylene oxide, solution 9, then once with absolute alcohol, solution 8, then to dump and dry.

Indicator lamps

95, 96 and 97 indicate as shown in FIG. 10. Lamp 94 indicates a waste tank full condition.

It may be mentioned that an alternate manifold arrangement has been employed experimentally with level sensing as distinct from positive metering. In the alternate arrangement the manifold allowed the solutions to flow by gravity and entry was made through a single port located at the top of the processing chamber, however, this was found not to offer the advantages of the present invention. The illustrated manifold of the'invention has proven particularly advantageous in providing a means for shortening flow passages and for minimizing intermixing of solutions. It also provides a means for mounting the solution control valves, the dump valve and the processing chamber valve as well as the metering pump. The use of a single solution entry and discharge port in the processing chamber reduces possible points of contamination from l0 to 1. Thus, while the method and apparatus of our own prior art worked and provided a highly significant advance in the art it can be seen that the present manifold arrangement has further advanced the art and in novel and significant respects.

Agitation and vibration of the solutions during processing to increase solution penetration through the receptacle pores has been tried but is generally not deemed necessary. Also, the required agitation or vibration apparatus has not been found to be justified by the results obtained. However, it is, of course, recognized that vibration or agitation may be employed and may in the future, as automatic tissue processing knowledge becomes more fully developed,prove justifiable in certain tissue techniques. Thus, a vibrator V, FIG. 8, may be employed to vibrate chamber 90 or a motor M, FIG. 8, may be employed to rotate rack 92 during processing somewhat like an electric ice cream freezer dasher to obtain solution agitation.

While not shown, the control panel and circuitry may include, depending on the specific program circuitry employed, a set switch to set the program logic circuitry once the external control switches have been selectively manually positioned and a reset switch to restore such logic circuitry to a particular program state. Such set and reset switches have been employed in one experimental embodiment and a starting sequence used which used the solution indicator lamps shown in FIG. 10 to indicate to the operator that a selected program has been set and is ready to start on a particular solution indicated by the associated solution indicator lamp, in solution indicator lamp bank 140.

What is deemed most significant and most important to providing a reliable day-to-day automatic electron microscopy processor is the achievement in this invention of a practical system of measuring nd minimizing fluid contamination. Also, of special importance to the present invention has been the achievement of a manual switch selector system that readily adapts to any of many well known forms of program circuitry to allow selection of base time, number of solution cycles, starting solutions, positive level measuring, particular solutions for particular processing and cleaning programs, and alternate terminal solutions for particular programs. The research investigator and histology technician is thus provided with a new-apparatus and method for both duplicating hand processing procedures and, more importantly, for allowing completely new processing techniques not heretofore known in the art.

While primarily directed to electron microscopy size tissue, it is recognized that much of the fluid exchange and timing apparatus of the invention lends itself to automatic slide tissue staining as welll as to light microscopy processing directed to processing 3 to 5 millimeter thick pieces of tissue. For staining slides the processing chamber of the invention may be in the form of a rectangular well and used in conjunction with a conventional staining slide tray adapted to hold, for example, 60 3 X 2 slides. The staining fluids would be recirculated rather than dumped but would be periodically changed depending on the amount of use. The solenoid valves and pump operation could be controlled accordingly and such staining could be performed at room temperature. For light microscopy work larger solution containers should be employed and all lines and pump surfaces handling paraffin should be maintained at a temperature high enough to keep the paraffin in a fluid state. The tissue receptacles for such ligth microscopy processing could be of the type shown in U.S. Pat. No. 3,411,185 and a plurality of such receptacles could be stacked in a perforated basket for moving into and out of the processing chamber. The processing chamber could be the same rectangular well form used for staining thus adding to the versatility. As with staining the fluids would normally be recirculated and selected fluids periodically changed depending on the amount of use.

I claim:

1. A closed processing system enabling a plurality of individual particles of minute electron microscopy size, having at least one dimension greater than 1 millimeter, to be separately contained while being bathed simultaneously for varying lengths of time in successive selected aqueous and non-aqueous electron microscopy tissue solutions so as to fix, dehydrate and clear the particles preparatory to embedding, comprising:

a. a pluraltiy 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 body portion defining an open cavity adapted to receive and retain a group of particles and closure means removably received by said body to enclose said cavity, the material forming said receptacles being inert to all of said solutions and having for each of said solutions at least some porous portion permeable thereto;

b. cabinet means providing a housing and respective refrigerated and non-refrigerated compartments therein;

c. a plurality of closed solution containers selectively mounted in said refrigerated and non-refrigerated compartments and each containing a particular electron microscopy tissue processing solution and collectively containing all of said aqueous and nonaqueous solutions;

d. a discharge pipe and an associated remotely and electrically controllable valve for each container connected to provide an individual selectively controllable flow path for each of said solutions to one side of its'respective corresponding valve, selected said pipes having at least a portion thereof positioned so as to be cooled in said refrigerated compartment;

e. a manifold structure fixedly mounted in said housing adjacent said compartments and mounting said container valves, said manifold structure providing about the periphery thereof an inlet port for and communicating with an opposite side of each respective said container valve, a centrally disposed, vertically directed passageway extending through said manifold, each said inlet port being communicated to said vertically directed passageway by a serparate one of a plurality of radially extending passageways, a pump port located in said periphery and a further radial passageway communicating said pump port with the vertical passageway;

f. a processing chamber mounted in said housing adjacent and above said manifold structure, said chamber having a removable top cover and being adapted to receive and process selected numbers of said receptacles simultaneously, said chamber having an entry-discharge port at the bottom thereof and a remotely and electrically controllable valve located between and controlling communication between said entry-discharge port and the top end of said vertical passageway;

g. remotely controllable metering pump means and positioned within said housing adjacent said manifold structure and having a pump inlet communi cating with said manifold structure pump port whereby said pump is enabled to withdraw and discharge through said pump port a positively measured volume of a selected said solution;

h. waste solution collection means including a waste discharge remotely electrically controllable valve communicating on one side with the bottom end of said vertical passageway and on an opposite side with a waste solution collector whereby when said waste valve is opened all solution fluids in said manifold passageways may flow to said waste collector for disposal; and

i. remotely operable electrical control means for remotely operating said container, chamber and waste valves and said pump means in a predetermined time sequence program whereby with a selected number of said receptacles installed in said chamber and said cover on said chamber said solutions are successively, selectively and independently drawn, externally measured, and admitted to said chamber in a predetermined volume, retained for predetermined times and drained from said chamber in a corresponding processing sequence such that selectedof said aqueous and nonaqueous solutions are exchanged with each said group of particles by penetration and drainage through said pores while maintaining said system closed and each said group physically isolated.

2. A system as claimed in claim 1 wherein said control means include adjustable timing means enabling said predetermined time of retention to be changed both with respect to all as well as selected ones of said solutions.

3. A system as claimed in claim 1 wherein said control means includes adjustable cycle switching means enabling selected solutions to be cycled more than once through said chamber.

4. A system as claimed in claim 1 wherein said control means includes time unit switching means enabling said predetermined time of retention to be varied with respect to selected said solutions.

5. A system as claimed in claim 1 wherein said control means includes hold-pass switching means enabling selected said solutions in one mode of switching to be by-passed and in another mode of switching to constitute a program terminal stage.

6. A system as claimed in claim 1 wherein said control means includes level selection switching means enabling a plural selection of volumes pumped into said chamber by said pump means.

7. A system as claimed in claim 1 wherein said control means includes switching means enabling any said solution to be a beginning stage in the said program.

8. A system as claimed in claim 1 wherein said control means includesprogram switching means adapted upon actuation to select certain said solutions and valves in a coordinated sequence adapted to flush selected said manifold and chamber surfaces for cleaning purposes.

9. A system as claimed in claim 1 wherein said manifold structure comprises a vertically disposed substantially cylindrical block containing said ports and passageways and having said container valves and pump means mounted on the side thereof, said chamber valve being mounted on an upper face and said waste valve on a lower face thereof. I i

10. A system as claimed in claim 1 having fan exhaust and fume collection means adapted to allow during operation of said system continuous withdrawal of solution fumes both from within said housing and from the area surrounding the top of said chamber.

11. A system as claimed in claim 1 wherein said control means includes:

a. adjustable timing means enabling said predetermined times of retention to be changed with respect to all said solutions,

b. adjustable cycle switching means enabling selected solutions to be cycled more than once through said chamber,

c. time unit switching means enabling said predetermined time of retention to be varied with respect to selected said solutions,

(1. hold-pass switching means enabling selected said solutions in one mode of switching to be by-passed and in another mode of switching to constitute a program terminal stage,

e. level selection switching means enabling a plural selection of volumes to be pumped into said chamber by said pump means,

f. switching means enabling any said solution to be a beginning stage in a program, and

g. switching means adapted upon actuation to select certain said solutions and valves in a coordinate subprogram sequence adapted to flush selected said manifold and chamber surfaces for cleaning purposes.

12. A system as claimed in claim 1 including means to vibrate said receptacle during processing.

13. A system as claimed in claim 1 including means to agitate the selected said solution in said chamber during processing.

14. A system as claimed in claim 1 wherein said chamber is mounted in said housing immediately adjacent said refrigerated compartment.

15. A system as claimed in claim 1 wherein said containers, pipes, valves, manifold structure, chamber and pump means are stationary within said housing.

16. A processing system for electron microscopy, light microscopy, staining and similar tissue processing enabling a plurality of individual tissue specimens to be bathed simultaneously for varying lengths of time in successive tissue solutions preparatory to other procedures, comprising:

a. holder means adapted to contain and physically isolate a plurality of said specimens from each other, the material forming said holder means being inert to all of said solutions and having for each of said solutions at least some portion permeable thereto;

b. cabinet means providing a housing and respective compartments therein;

c. a plurality of closed solution containers mounted in said housing and each containing a particular tis sue processing solution;

d. a discharge pipe and an associated remotely and electrically controllable valve for each container connected to provide an individual selectively controllable flow path for each of said solutions to one side of its respective corresponding valve;

e. a manifold structure fixedly mounted in said housing adjacent said compartments and mounting said container valves, said manifold structure providing about the periphery thereof an inlet port for and communicating with an opposite side of each respective said container valve, a centrally disposed, vertically directed passageway extending through said manifold, each said inlet port being communicated to said vertically directed passageway by a separate one of a plurality of radially extending passageways, a pump port located in said periphery and a further radial passageway communicating said pump port with the vertical passageway;

f. a processing chamber mounted in said housing adjacent and above said manifold structure, said chamber having a removable top cover and being adapted to receive said holder means and to process selected numbers of said specimens simultaneously, said chamber having an entry-discharge port at the bottom thereof and a remotely and electrically controllable valve located between and controlling communication between said entrydischarge port and the top end of said vertical passageway;

g. remotely controllable metering pump means positioned within said housing adjacent said manifold structure and having a pump inlet communicating with said manifold structure pump port whereby said pump is enabled to withdraw and discharge through said pump port a positively measured volume of a selected said solution; and

h. remotely operable electrical control means for remotely operating said container and chamber valves and said pump means in a predetermined time sequence program whereby with a selected number of said specimens installed in said chamber and said cover on said chamber said solutions are successively, selectively and independently drawn, externally measured, and admitted to said chamber in a predetrmined volume, retained for predetermined times and drained from said chamber through valvular means connected to said vertical passageway in a corresponding processing sequence such that selected of said solutions are exchanged with each said specimen while maintaining said system closed and each said specimen physically isolated from the other.

17. A system as claimed in claim 16 wherein said manifold structure comprises a vertically disposed substantially cylindrical block containing said ports and passageways and having said container valves and pump means mounted on the side thereof.

18. A system as claimed in claim 17 having fan exhaust and fume collection means adapted to allow during operation of said system continuous withdrawal of solution fumes both from within said housing and from the area surrounding the top of said chamber.

Patent No. 3 l7l ,490 Dated November 13 1973 Inventor(s) Thomas D. Kinney and John E. -P. Pickett 7 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. line 41., "3,536,203" should be 3,526,203-.

Col. line 27, "switching" should be -switch-.

Col. line 64, "fam" should be fan-.

line 53; "ligth" should be -light.

Col. 6, line 54, "inention" should be -invention Col. 9, I l

Col. line 65, no should be inserted after "dimension" Col. 10, line 4, "plureltiy" should be -plurality-. Q Col. 10, line 56, "and" after "means" should be deleted.

Col. 14, line 4, "predetrmined" should be -predetermined-,

Signed and sealed this 6th day of August 1974.

I. (SEAL) Attest: I MCCOY M. GIBSON, JR. c. MARSHALL DANN Attesting Officer Commissioner of Patents FORM PO-IOSO (10-69) USCOMM-Dc 60376-P69 U45 GOVERNMENT PRINTING OFFICE: "i9 O-Ji6-33l,

Claims

1. A closed processing system enabling a plurality of individual particles of minute electron microscopy size, having at least one dimension greater than 1 millimeter, to be separately contained while being bathed simultaneously for varying lengths of time in successive selected aqueous and non-aqueous electron microscopy tissue solutions so as to fix, dehydrate and clear the particles preparatory to embedding, comprising: a. a pluraltiy 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 body portion defining an open cavity adapted to receive and retain a group of particles and closure means removably received by said body to enclose said cavity, the material forming said receptacles being inert to all of said solutions and having for each of said solutions at least some porous portion permeable thereto; b. cabinet means providing a housing and respective refrigerated and non-refrigerated compartments therein; c. a plurality of closed solution containers selectively mounted in said refrigerated and non-refrigerated compartments and each containing a particular electron microscopy tissue processing solution and collectively containing all of said aqueous and non-aqueous solutions; d. a discharge pipe and an associated remotely and electrically controllable valve for each container connected to provide an individual selectively controllable flow path for each of said solutions to one side of its respective corresponding valve, selected said pipes having at least a portion thereof positioned so as to be cooled in said refrigerated compartment; e. a manifold structure fixedly mounted in said housing adjacent said compartments and mounting said container valves, said manifold structure providing about the periphery thereof an inlet port for and communicating with an opposite side of each respective said container valve, a centrally disposed, vertically directed passageway extending through said manifold, each said inlet port being communicated to said vertically directed passageway by a serparate one of a plurality of radially extending passageways, a pump port located in said periphery and a further radial passageway communicating said pump port with the vertical passageway; f. a processing chamber mounted in said housing adjacent and above said manifold structure, said chamber having a removable top cover and being adapted to receive and process selected numbers of said receptacles simultaneously, said chamber having an eNtry-discharge port at the bottom thereof and a remotely and electrically controllable valve located between and controlling communication between said entry-discharge port and the top end of said vertical passageway; g. remotely controllable metering pump means and positioned within said housing adjacent said manifold structure and having a pump inlet communicating with said manifold structure pump port whereby said pump is enabled to withdraw and discharge through said pump port a positively measured volume of a selected said solution; h. waste solution collection means including a waste discharge remotely electrically controllable valve communicating on one side with the bottom end of said vertical passageway and on an opposite side with a waste solution collector whereby when said waste valve is opened all solution fluids in said manifold passageways may flow to said waste collector for disposal; and i. remotely operable electrical control means for remotely operating said container, chamber and waste valves and said pump means in a predetermined time sequence program whereby with a selected number of said receptacles installed in said chamber and said cover on said chamber said solutions are successively, selectively and independently drawn, externally measured, and admitted to said chamber in a predetermined volume, retained for predetermined times and drained from said chamber in a corresponding processing sequence such that selected of said aqueous and non-aqueous solutions are exchanged with each said group of particles by penetration and drainage through said pores while maintaining said system closed and each said group physically isolated.

8. A system as claimed in claim 1 wherein said control means includes program switching means adapted upon actuation to select certain said solutions and valves in a coordinated sequence adapted to flush selected said manifold and chamber surfaces for cleaning purposes.

9. A system as claimed in claim 1 wherein said manifold structure comprises a vertically disposed substantially cylindrical block containing said ports and passageways and having said container valves and pump means mounted on the side thereof, said chamber valve being mounted on an upper face and said waste valve on a lower face thereof.

11. A system as claimed in claim 1 wherein said control means includes: a. adjustable timing means enabling said predetermined times of retention to be changed with respect to all said solutions, b. adjustable cycle switching means enabling selected solutions to be cycled more than once through said chamber, c. time unit switching means enabling said predetermined time of retention to be varied with respect to selected said solutions, d. hold-pass switching means enabling selected said solutions in one mode of switching to be by-passed and in another mode of switching to constitute a program terminal stage, e. level selection switching means enabling a plural selection of volumes to be pumped into said chamber by said pump means, f. switching means enabling any said solution to be a beginning stage in a program, and g. switching means adapted upon actuation to select certain said solutions and valves in a coordinate subprogram sequence adapted to flush selected said manifold and chamber surfaces for cleaning purposes.

16. A processing system for electron microscopy, light microscopy, staining and similar tissue processing enabling a plurality of individual tissue specimens to be bathed simultaneously for varying lengths of time in successive tissue solutions preparatory to other procedures, comprising: a. holder means adapted to contain and physically isolate a plurality of said specimens from each other, the material forming said holder means being inert to all of said solutions and having for each of said solutions at least some portion permeable thereto; b. cabinet means providing a housing and respective compartments therein; c. a plurality of closed solution containers mounted in said housing and each containing a particular tissue processing solution; d. a discharge pipe and an associated remotely and electrically controllable valve for each container connected to provide an individual selectively controllable flow path for each of said solutions to one side of its respective corresponding valve; e. a manifold structure fixedly mounted in said housing adjacent said compartments and mounting said container valves, said manifold structure providing about the periphery thereof an inlet port for and communicating with an opposite side of each respective said container valve, a centrally disposed, vertically directed passageway extending through said manifold, each said inlet port being communicated to said vertically directed passageway by a separate one of a plurality of radially extending passageways, a pump port located in said periphery and a further radial passageway communicating said pump port with the vertical passageway; f. a processing chamber mounted in said housing adjacent and above said manifold structure, said chamber having a removable top cover and being adapted to receive said holder means and to process selected numbers of said specimens simultaneously, said chamber having an entry-discharge port at the bottom thereof and a remotely and electrically controllable valve located between and controlling communication between said entry-discharge port and the top end of said vertical passageway; g. remotely controllable metering pump means positioned within said housing adjacent said manifold structure and having a pump inlet communicating with said manifold structure pump port whereby said pump is enabled to withdraw and discharge through said pump port a positively measured volume of a selected said solution; and h. remotely operable electrical control means for re-motely operating said container and chamber valves and said pump means in a predetermined time sequence program whereby with a selected number of said specimens instaLled in said chamber and said cover on said chamber said solutions are successively, selectively and independently drawn, externally measured, and admitted to said chamber in a predetrmined volume, retained for predetermined times and drained from said chamber through valvular means connected to said vertical passageway in a corresponding processing sequence such that selected of said solutions are exchanged with each said specimen while maintaining said system closed and each said specimen physically isolated from the other.