JPH0122573B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a specimen preparation device for creating permanent optically transparent specimens for cytology, histology and pathology research mounted on microscope slides.

In the field of medicine, it is common practice to prepare thin sections of biological specimens on glass slides for microscopic studies. Such sections are colored (to increase clarity for inspection);
Dehydrate and perform fixation treatment (specimen preparation treatment). Generally, such fixation procedures confine each individual specimen within a medium supported by a microscope slide and hold the specimen therein for an indeterminate amount of time. For this purpose, a solvent solution of a rigid synthetic resin is used as the mounting medium or mountant. Such resins are useful because they (i) do not fade the color of the colorant used to treat the specimen, (ii) exhibit only a slight yellowing even with age, and (iii) maintain the mean refractive index of the tissue (1.530 to 1.570) after drying. ) is chosen to have a refractive index very close to . For example, with a fixative having a refractive index close to that of the specimen, almost complete transparency can be obtained. β-pinene resin is often used as a fixative. To learn about other available fixatives, please visit Histopathic Techniques and Plastics Histochemistry.
Technique and Practical History (Chemistry)
Please refer to Table 5-1 on page 98.

Generally, fixatives are dissolved in a volume of solvent to a viscosity suitable for dispensing a controlled volume over the specimen. To create the flat top surface necessary for proper microscopy, it is common to place a glass cover piece over the dispensed fixative while it is still liquid. Each microscope slide undergoes an extended drying process, which may require five to six days or more, to remove any solvents that may be present that would interfere with the testing process. During such processing, each microscope slide is placed in a horizontal position and optionally in a mild environment to facilitate evaporation of the solvent. Evaporation of such solvents (i) hardens the fixative;
(ii) It is necessary to seal the cover piece to allow handling of each slide and (iii) to avoid optical interference due to the low refractive index of the residual solvent during examination.

Currently, fixation and drying treatments are performed artificially in the laboratory. Generally, after removing the microscope slide from the so-called clean solution and quickly expelling this solution, one to several drops of fixative are deposited onto the specimen by the operator. The amount of such fixative must be sufficient to occupy the space between the cover piece and the specimen when applied. The operator then gently lowers the cover piece onto the fixative and carefully positions it. In this method, one side of the cover strip is placed over the fixative, the fixative is then released, and a portion of the fixative is placed in front of the cover strip as it is lowered into position. to press. The cover piece was slightly adjusted by the operator to form a thin, even fixative coating.
Remove any trapped air bubbles from the underside of the cover piece.
It is necessary to properly center the cover piece over the specimen. Any excess fixative forced out of the cover piece must be quickly and carefully removed by the operator, for example with blotting paper.

The most common failures associated with fixation of specimens are (i) delay in applying the fixative after removing the specimen from the detergent, which causes the specimen to dry out and cause some deterioration of the tissue structure; (iii) removing the cover piece too quickly or inadvertently; If applied to the fixative, it will trap air bubbles and these air bubbles will become increasingly difficult to release as the fixative begins to dry.

The disadvantages of the conventional methods briefly mentioned are well recognized. Among these disadvantages are:

(i) Fixatives must be applied carefully and quickly by the operator, as the specimen will be damaged if it dries.

(ii) Since the solvent in the fixative has a low refractive index and only slowly diffuses out from the underside of the cover piece during the drying process, the fixative is generally sufficient to permit normal dispensing with a minimum of solvent. Just mix it up. However, if the application of the cover piece is delayed, the surface of the fixative will begin to dry, increasing the risk of trapping air bubbles on the underside of the cover piece. The cover piece must therefore be placed carefully and quickly on the fixative.

(iii) If excess fixative is dispensed, such excess must be squeezed out from the periphery of the cover piece. In order to properly remove such excess, it is necessary to use a suitable solvent. This solvent flows down the underside of the cover piece and removes the fixative underneath, leaving an air space that ultimately interferes with the test.

(iv) During the drying process, the slides must be placed laterally on a flat horizontal surface for a significant period of time. Tilting the slide during such processing often causes the cover strip to slip away from the tissue section. Also, since the periphery of the fixative extends slightly beyond the periphery of the cover strips and the diffusion of solvent from the underside of such cover strips makes these peripheries tacky, each slide should be kept dry at the end of the drying process. cannot be stacked for effective storage.

(v) A minimum amount of solvent remains under the central part of the cover piece even after a long drying process. Because the solvent has a relatively low refractive index, the optical properties of the slide are suboptimal over long periods of time.

(vi) If the tissue specimen has high spots, a thick layer of fixative is present on the underside of the cover piece. The drying process is therefore slow and the fixative shrinks significantly as the solvent evaporates. Such shrinkage often occurs unevenly and locally, creating air spaces beneath the cover piece that obscure tissue details.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a device for fixing tissue specimens so that the overall processing time is significantly shortened.

Furthermore, it is an object of the present invention to fix tissue specimens and substantially eliminate the long drying time in conventional methods;
The present invention seeks to provide a novel device that allows mechanically stable, dry, fixed specimens to be readily available for testing.

Another object of the present invention is to provide an apparatus that can quickly prepare an optically transparent and stable specimen having a refractive index very close to that of the specimen.

According to the invention, a fixative consisting of a mixture of a low volatility, low viscosity liquid acrylic reactant and a UV sensitive catalyst system is used as the final encapsulation medium. Such fixatives retain their free-flow properties until polymerized, which then results in a hardened, optically clear, solvent-impermeable, solvent-resistant medium that completely encapsulates the specimen. Such a fixing agent does not cause the color of the coloring agent to fade and maintains a colorless and transparent state. The acrylic reagent is specifically selected to provide a final refractive index of 1.530 to 1.570 (typically 1.550) after polymerization to match the refractive index of the specimen. As is well known, the refractive index can be determined by, for example, mixing two components with a lower refractive index and a higher refractive index and changing the ratio so that an arbitrary intermediate refractive index can be obtained. can be controlled.

Since dehydrated sections must not be allowed to dry, specimen-supporting slides are first immersed in a low-viscosity, high-volatility solvent solution of a low concentration of fixative. Such a fixative may be made of the same material as the final encapsulation medium or a corresponding compatible material so that the same refractive index is obtained after polymerization. Next, dry the liquid on the slide. Unlike standard methods, all the solvent is evaporated, and a very thin protective layer of solvent-free liquid fixative now remains on the specimen to prevent it from drying out. Since the components of such fixatives have very low volatility, they do not evaporate significantly. The tissue is then kept moist and penetrated by the fixative. Once the solvent has evaporated, usually within one minute, a suitable final volume or layer of final encapsulating fixative is applied to the wet section and the cover strip is gradually applied. The fixative is then exposed through the cover strip (or microscope slide) to ultraviolet light, for example from a low wattage fluorescent black light lamp, causing the fixative to polymerize within one minute. Therefore, the subsequent fixative layers applied to the underside of the cover piece become integral and fully cured. Furthermore, the resulting fixative contains almost no solvent that would interfere with the testing process, and thus can be tested immediately.

It is also an advantage of the present invention that the slight excess fixative extruded from the underside of the cover piece remains liquid as it remains exposed to atmospheric oxygen. This oxygen acts as a polymerization inhibitor for the thin reactant layer, as is well known. Therefore, the microscope slide can be thoroughly washed with a suitable solvent to remove excess liquid fixative extruded from the cover piece without affecting the polymerized fixative, or wiped dry by the operator for immediate examination. so that it can be stored or stacked for storage.

Therefore, the present invention generally involves doubly applying the fixative to the specimen. The first application of fixative is done with fixative and volatile solvent immediately after removal of the microscope slide from the wash solvent, and the subsequent application is done with pure fixative. Each such fixative contains a polymerizing agent, preferably UV sensitive and compatible with other fixatives. During initial application, the solvent is sufficiently evaporated from the uncoated fixative to protect the specimen from drying out and allow the liquid fixative to penetrate. 2
During the second application, any additional required volume of fixative is applied over the first fixative layer and then the cover strip is applied and both layers polymerized simultaneously. The separately applied fixative layers are thus brought together and all solvent is sufficiently removed to permit inspection immediately after the polymerization operation.

Embodiments of the specimen preparation device according to the present invention will be described in detail below with reference to the accompanying drawings.

The apparatus shown in FIG. 1A is the specimen preparation apparatus of the present invention. This specimen preparation device includes a feeding device 1 that sequentially delivers microscope slides 3 each supporting a tissue specimen to be fixed for examination. Each microscope slide 3 has a corresponding tissue specimen 5
It is provided with a label part 7 for identifying. First, each tissue specimen 5 is placed on a corresponding microscope slide 3 in the usual manner, dried, paraffin removed, and
Water is added, colored, dehydrated, cleaned, and then immersed in a liquid consisting of a low concentration (1% to 25%) fixative solution in a low viscosity, high volatility solvent. Such liquid is contained in a container 9, as shown in FIG. 1B. Each microscope slide 3 is immersed into the container 9 by the operator immediately after a normal cleaning process to leave the specimen moist with a volatile solvent such as xylene. The liquid in container 9 consists of, for example, xylene, toluene or Freon TF and a suitable fixative. This fixative is a fixative that consists of a mixture of a low volatility, low viscosity liquid acrylic reactant and a UV sensitive catalyst system and becomes transparent upon polymerization.
Upon immersion into the container 9, a thin liquid layer forms on the tissue specimen 5 after removal. The solvent in such a liquid is sufficiently evaporated so that a very thin layer of solvent-free liquid fixative remains on both sides of the microscope slide 3. In this case, the tissue specimen 5 remains moist and the fixative penetrates throughout. Each microscope slide 3 is placed on the feeder 1 one after the other and sent to the layering unit 11 one after the other.

Layering unit 11 serves to form a second layer on tissue specimen 5 consisting of the same fixative as described above. The fixatives include low volatility, low viscosity liquid acrylic reagents such as monomers or oligomers of methacrylic esters, acrylic esters and vinyl compounds and benzoyl, benzoin ether, acetophenone or Michler's ketone. It consists of a mixture with a UV-sensitive catalyst and becomes transparent when polymerized. Furthermore, the layered unit 11 exposes the fixing agent layer to ultraviolet radiation that peaks near the absorption maximum of the catalyst so that both layers polymerize together. Subsequently, each microscope slide 3 is passed through a cleaning device 13 one after the other. A cleaning device 13 cleans the surface of each microscope slide 3 to remove all unpolymerized fixative remaining thereon. Each cleaned microscope slide 3
is subsequently passed through a drying device 15 to accelerate the evaporation of the washing liquid, and then to a stacking device 17 to accumulate fully processed microscope slides 3. Each of these microscope slides 3 is immediately available for examination. The total processing time required to sufficiently fix, wash and dry each microscope slide 3 from its introduction into the layered unit 11 is approximately 2 minutes.

To describe this specimen preparation device in more detail, each microscope slide 3 carrying a tissue specimen 5 is
After being manually dipped into the container 9 and taken out from the container 9, the operator manually places it on the feeding device 1. Although it has been described that the microscope slide 3 is immersed and positioned manually, the present invention can also provide a structure as an integral part of the feeding device 1 to directly perform this operation. The feeding device 1, only partially illustrated in FIG. 1A, comprises a parallel belt system consisting of endless belts 19, 21 and suitably driven rollers 23. The rollers 23, together with cooperating idler rollers (not shown), are recessed along the central surface to form shoulders on which the endless belts 19, 21 are respectively hung. Each microscope slide 3 is attached to an endless belt 19 parallel to each other.
21, and the tissue sample 5 is placed on the bottom surface between the endless belts 19 and 21. Furthermore, a pair of mutually parallel guides 55, only one of which is illustrated, is positioned adjacent to each of the endless belts 19, 21 to permit proper positioning and orientation of the microscope slide 3 relative thereto.

The passage of each microscope slide 3 along the transport device 1 is detected by a feeler which initiates the operation of the specimen preparation device according to the invention in relation to the processing of each such microscope slide. Such a feeler is connected to the rotating shaft 27
It is provided with a finger piece 25 attached to one end. The rotating shaft 27 is supported by a bearing 28, and fingers 25 extend downward through the space between the endless belts 19, 21. The passage of the microscope slide 3 pushes the finger piece 25 and rotates the rotating shaft 27, which actuates the micro switch 29.

When actuated, microswitch 29 initiates operation of lamination unit 11 as described above. stratigraphic unit 1
1 comprises a horizontal push plate piece or support piece 31 arranged between each idler roller 35,37. A continuous optically smooth ribbon 33 is pulled along each roller 35,37. The ribbon 33 is supplied from a supply winding frame 39 and is advanced by a winding winding frame 41. The ribbon 33 has idle rollers 36 and 38.
Maintain appropriate tension. The operation of the winding frame 41 is started by the operation of the micro switch 29.
Further actuation of microswitch 29 initiates the dispensing of fixative onto top surface 43 of ribbon 33 for final encapsulation of tissue specimen 5 in microscope slide 3. The microscope slide 3 has now been advanced into the layered unit 11 .

A dispenser 45 is located above and adjacent the top surface 43 of the ribbon 33 and is connected via a valve 47 and along a conduit 49 to a source of pressurized fixative 51 . Valve 47 is activated by closing microswitch 29 after a suitable delay time to dispense a continuous bead 53 of fixative onto surface 43 of ribbon 33. The length of the bead 53 is preferably the same as the length of the microscope slide. The fixative is also dispensed in such a proportion that a thin layer of fixative is ultimately formed between the opposing surfaces of the microscope slide 3 and the ribbon 33 as described above.

Each microscope slide 3 is attached to a feeder 1 as shown.
From above each of the endless belts 19, 21, only one of them is sequentially advanced to the first inclined table having the illustrated guide 55. Each guide 55 is mirror-image L-shaped and forms a shoulder part for supporting the microscope slide 3 during passage and a guide part for orienting the microscope slide 3. The slope of the guide 55 is such that gravity feeds each microscope slide 3 onto the ribbon 33.
Advancement of the ribbon 33 places the microscope slide 3 on a bead of fixative 53 dispensed onto the surface of the ribbon 33. When the ribbon 33 first contacts the front end of each microscope slide 3, such overlap of the microscope slides forces the fixative to the periphery, spreading the fixative between all mutually opposing surfaces of the microscope slide 3 and the ribbon 33. In this case, the ribbon 33 acts like a cover piece in a conventional fastening method. The volume of fixative applied to ribbon 33 should be only slightly in excess of that necessary to ensure the formation of a continuous layer of fixative between such opposing surfaces. The active spreading of the fixative between the opposing surfaces of the microscope slide 3 and the ribbon 33 and the flattening of the ribbon 33 ensure that irregularities in the surface of the first layer of fixative are filled. Also, the resulting or finished surface of the fixative is optically smooth.

When the ribbon 33 is further advanced by the winding spool 41, the microscope slide 3 is conveyed through the chamber 57. Chamber 57 is equipped with a suitable black light lamp (not shown) to polymerize the fixative layer between the mutually opposing surfaces of microscope slide 3 and ribbon 33. Such a lamp is energized by the microswitch 29 together with the energization of the winding frame 41. In the chamber 57, the fixative layer placed between the ribbon 33 and the microscope slide 3 is fully cured.

As shown, the direction of the ribbon 33 is reversed after passing around rollers 37 by providing a tension roller 38 to peel the ribbon 33 from the surface of the newly polymerized fixative and collect it in a winding frame 41. do. The newly exposed surface of the fixative encapsulating the tissue specimen 5 is optically smooth because it is bounded by the ribbon 33. However, the protective layer deposited on each surface of the slide 3 with a slight excess of fixative extruded from between the ribbon 33 and the microscope slide 3 will not react unless its polymerization is inhibited by exposure to atmospheric oxygen. I don't. Each microscope slide 3 is then mirror-imaged into each L-shaped guide 5.
Proceed to the second ramp with 9. Only one of each guide 59 is shown and is constructed similarly to guide 55. The microscope slide 3 is fed by gravity along a guide 59 to a moving parallel belt combination consisting of mesh belts 61, 63. Each mesh belt 61, 63 rests on a shoulder formed at the mutually opposite ends of a suitable driven roller 65 and a corresponding idler roller 67. The center surface of each roller 65, 67 is preferably recessed. The driven roller 65 is
It is preferable to drive the microscope slide 3 to the guide 59 by the microswitch 29 after a suitable delay time.

The cleaning device 13 includes a chamber 69 in which holes are formed in wall portions 71 and 73 facing each other so as to receive mesh belts 61 and 63, which are endless belts. The portion 75 of each guide 55 is connected to the mesh belt 6
During the passage of the chamber 69 along the lines 1, 63, the mesh belt 61,
Maintain the orientation of the microscope slide 3 on the 63. A suitable washing fluid such as Freon TF to remove unreacted fixative from the surface of the microscope slide 3.
is supplied from the container 77. Although not shown, container 7
7 is equipped with a positive displacement pump which is energized by a micro switch 29 at the same time as the driven roller 65.
The outlet of this pump is connected to a conduit 79 which is connected to two branch pipes 81,83. Each branch pipe 81, 8
The outlets of 3 are arranged to direct pumped cleaning fluid to the lower and upper surfaces of the microscope slide 3, respectively, as it passes through the chamber 69. The outlet of each branch 81, 83 is configured to direct a spray approximately perpendicular to the lower and upper surfaces of the microscope slide 3, respectively. The cleaning liquid subsequently collects at the bottom of chamber 69 and enters container 77 for circulation along drain pipe 85 and conduit 87.

When the microscope slide 3 exits the chamber 69, the slide 3 is moved to each blower 8 by each mesh belt 61, 63.
Carried between 8 and 89. Each blower 88, 89 is energized together with a driven roller 65 and a pump housed in a container 77. Each blower 88 , 89 directs warm air onto opposing surfaces of the microscope slide 3 to effectively evaporate any residual cleaning liquid remaining on the microscope slide 3 . The fixation of the tissue specimen 5 on the microscope slide 3 is thus completed, and this microscope slide is immediately available for examination.

Following the drying process, the microscope slides 3 are fed from each mesh belt 61, 63 into the stacking device 17.
The stacking device 17 includes a box 91 provided with an integral inclined portion 92 so as to receive each microscope slide 3 sent one after another from each mesh belt 61, 63.
The end of each guide 55 cooperates with a ramp 93 in maintaining the proper orientation of each such microscope slide 3. Box 91 contains microscope slide 3 to be sent to this box.
A false bottom 97 is provided which is supported on spring pieces 99 to minimize the fall of the base. Box 91 has a structure with four sides. Two wall sections have been removed to illustrate the stack of accumulated microscope slides 3. Since the fixative encapsulating the tissue specimen 5 on each microscope slide 3 is sufficiently reacted and dry, stacking each such microscope slide 3 immediately will not affect its optical properties or Do not allow the slides to stick together.

The specimen preparation device shown in FIG. 1A has been described for fixing tissue specimens onto separate microscope slides, but when each of the above-mentioned components controlled by the microswitch 29 passes an individual microscope slide through this specimen preparation device, It is clear that these microscope slides can be operated for a sufficient time to individually process these microscope slides. Each of these same parts is the feeding device 11
It can, of course, be operated continuously for fixation of tissue specimens that are automatically directed one after the other along the line.

Although the present invention has been described in detail with reference to its embodiments, it is obvious that the present invention can be modified in various ways without departing from its spirit.

Effects of the Invention According to the present invention, when mounting a biological specimen on a microscope slide, it is possible to protect the biological specimen from drying out and prevent its deterioration, and also to prevent the surface of the first layer of the fixative from being damaged. It ensures that the regular areas are filled with a second layer of fixative, resulting in an optically smooth fixative surface, and further allows for sequential application of double layers of fixative to biological specimens on multiple microscope slides. can be automatically formed.

[Brief explanation of drawings]

FIG. 1A is a perspective view of one embodiment of the specimen preparation device of the present invention for fixing a tissue specimen to a microscope slide;
Figure B is a vertical sectional view of an auxiliary device used in cooperation with the specimen preparation device of Figure 1A. 1... Feeding device, 3... Microscope slide, 5...
... Specimen, 9... Container, 11... Stratigraphic unit, 33...
...Ribbon, 45...Dispensing device, 57...Chamber.

Claims (1)

[Claims] 1. A specimen preparation device for preparing a plurality of specimens attached to a microscope slide, comprising: (a) a plurality of microscope slides each having a first surface facing in the same direction; (c) a plurality of separate thin biological cytoplasmic specimens mounted on said first surface of a microscope slide; (d) a first material dispensing device adapted to be immersed in a neutral solvent; (e) a second material dispensing device for further immersing the specimen in an amount of the polymerizable material sufficient to encapsulate the specimen; and (f) the microscope slide. , after evaporation of the volatile solvent, a second feeding device adapted to sequentially convey the material into a properly aligned state for cooperation with the second material dispensing device; (h) a continuous, elongated, flat ribbon that is smooth and impermeable to oxygen; (h) an apparatus suitable for applying the ribbon onto said encapsulated specimen; To,
A specimen preparation device comprising: an exposure device for exposing the polymerizable material to ultraviolet light; and (v) a removal device suitable for removing the ribbon from the encapsulated specimen. 2 further disposed along the path carrying said microscope slides and adapted to remove any non-polymerized material by sequentially washing said microscope slides when said specimen preparation device is activated; A specimen preparation device according to claim 1, comprising a cleaning device 13 configured as follows. 3. A drying device 15 further arranged along said path and configured to be suitable for sequentially drying said microscope slides after washing when said specimen preparation device is activated. Specimen preparation device according to scope 2. 4. A stacking device 17 further arranged along said path and configured to be suitable for stacking each of said plurality of microscope slides in sequence, after drying, when said specimen preparation device is activated. A specimen preparation device according to claim 3.