Paper - The use of guide planes and plaster of Paris for reconstructions from serial sections

Embryology - 18 May 2024        Expand to Translate
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Lewis WH. The use of guide planes and plaster of Paris for reconstructions from serial sections- Some points on reconstruction. (1915) Anat. Rec. :719-

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This historic 1915 paper by Lewis describes research techniques employed to generate models of the human embryos in the Carneige Collection. Modern Notes: Carneige Collection

The Use Of Guide Planes And Plaster Of Paris For Reconstructions From Serial Sections: Some Points On Reconstruction

Warren H. Lewis

From the Anatomical Laboratory, Johns Hopkins University

Five Figures

Owing to the generosity of the Carnegie Institution of Washington, funds were made available for reconstructing the head of a 21 mm. human embryo, No. 460, Mall collection. During the process of this reconstruction, some rather helpful methods were developed, which others who are engaged in similar work may find useful, and with this in view I have been urged to publish the methods employed.

In regard to the preservation and staining of the embryos, as well as the cutting and mounting, it is assumed that these operations have been carried out in such a manner that the series is practically perfect, and that no unavoidable shrinkage has occurred during the dehydration and embedding; and that in cutting the orientation is such as to give as nearly perfect horizontal, sagittal or frontal sections as is possible. Great care must be taken in mounting the sections on the slides in order to avoid distortions. Reconstruction is undoubtedly greatly facilitated by the use of guide lines in the sections, such as are produced by the ordinary camphor black method. Unfortunately, such guide marks were not present on any of the series of sections used and it was necessary to depend for the form on photographs or camera drawing of the whole embryo, made before cutting. These should be as nearly as possible from lateral, frontal at horizontal views.

Photographs Of Sections

I have been able to abandon the laborious and time—consuming method of drawing or tracing each section projected in the usual manner onto paper, and have substituted the more expensive but far better method of photographing on large plates, and using the line bromide or azo G hard (matte) prints. While this method is more expensive as regards the immediate outlay of money, it is much cheaper in the end than the old method of tracing, when account is made of the time involved. The photographs are far superior to any drawing that can possibly be made and greatly facilitate the work, both on account of the greater accuracy and the greater wealth of detail. The various structures to be reconstructed may first be colored on the photograph, thus enhancing the clearness and sharpness of the picture. The ordinary Sussner creta polycolor pencils were used. The photographs of the sections were made in a dark—room with the ordinary Zeiss projection apparatus and for ordinary diameters, 40 or 50, the Zeiss 5 cm. planar lens was used. This is an ideal lens for such work since there is no measurable distortion of the image. In the place of an ordinary are lamp, we substituted-a 250—watt mazda stereopticon bulb, a round bulb with filaments grouped together in a small ball at the center. To avoid unequal illumination from the spiral filaments a ground glass plate was interposed directly iI1 front of the light. The advantages of the mazda light over the are are (1) it remains constant. and (2) it can easily be turned on or off for time exposures.

The arrangement of the Zeiss optical bench is shown by the diagram, figure 1. I am aware that this may not be the most perfect arrangement, still we were able to obtain excellent results. The most important point consists in focusing each section by moving the slide carrier back and forth, the lens remaining fixed, instead of the usual method of moving the lens back and forth. When the magnification is once adjusted to the required diameter, the lens (7) is securely fixed in position and the plate—holder (14) likewise. Thereafter, the object or slide is brought into focus by means of a fine adjustment connected with the focusing—rod (10). Magnification is thus not altered from section to section or from slide to slide, since the sections are thus brought in the focus of the lens. This insures equal magnification of every section, the most important condition for accurate reconstruction. This method of focusing was introduced into the laboratory by Doctor Essick.

The dark-room by this method becomes the camera, in which a perpendicular board (14) with slots for the plates takes the place of the plate—holder. This board is pivoted in the center and can be freely turned at any angle in a perpendicular plane and clamped there by means of a thumb—screw at the back.

The plate—holder—board is attached to -a movable stand, which can be moved to or from the lens in a straight line only, and can be securely clamped in position when in the proper place. At 50 diameters’ magnification with the 50 mm. planar lens the plate—holder should be about 6 ft. and 8 in. from the lens. A series of holes can be so placed on the plate-board, as at a, b, c, figure 1, into which the clamp bolt can be changed and the board given a new center for different sized plates. An old plate having the same thickness as those about to be exposed was used for focusing, after having had a fine piece of white paper tightly pasted over it. An undeveloped plate is still better.

Fig. 1 Arrangement of Zeiss optical bench for photographing: 1, 150-watt mazda stereopticon bulb; 2, groundglass plate; 3. 5-in. concave, convex condensing lens; 4, 5-in.p1ano-convex lens; 5, condensing lens; 6‘, mechanical slide carrier; 7. 50 mm. Zeiss planar lens; 8, light-proof hood over entire bench with curtain on operating side; .9, optical bar, distances between the feet of the various lens. etc., shown in inches and fractions; 13 movable stands; 1./;,_‘pla.te holder board; 15, plate.

Standard orthonon and Stanley commercial plates were used. The latter are apparently just as good and much cheaper than the former. An exposure of about 5 seconds gave the best results; diaphragm of the planar ‘lens at 4. Flat negatives with very little contrast give the best prints, while ‘contrasty’ plates, which appear very beautiful, give very poor prints since the shadows and high lights are in too strong contrast. When the shadows and high lights are in strong contrast in the sections—— as was the case in the series used, since the central nervous system was deeply stained with alum cochineal and the delicate connective tissue but faintly stained—it is not easy to get negatives which will give prints showing detail in both regions. With full development, a strong light and short exposure give flatter, softer negatives than a dim light and long exposure. The diaphragm of the lens should be as wide open as is consistent with a sharp image, to increase light and reduce time of exposure. It is important to use a developer that will help to give softness to the negative and the following formula is recommended. There is a decrease in the amount of sodium carbonate usually employed for the purpose of increasing the softness of the negative:

Formulae for pyro developer

Solution No. 1 Solution No. 2 Solution No. 3. Water.... . . . . .470 cc. Water.......... . .470 cc. Water . . . . . . . . . . . .470 cc. Oxalic a.cid....700 mg. Sod. sulph.... . . 60 gms. Sod. carb..... . . .. 60 gms.

Pyrogal. acid. 30 gms.

For tank development use 30 cc. each of solutions 1, 2, 3, to 1000 cc. water; develop 20 minutes at 70° F. For tray use 60 cc. each of solutions 1, 2, 3, to 1000 cc. water; develop 5 minutes at 70° F.

Add 1 drop of a 10% solution of Potassium Bromide to every 30 cc.

l"r".'c'irLg bath formula

Hypo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 480 gms. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..1920 cc.

With acid hardener

Water . . . . . . . . . . . . . . . . . . . . . . .150 cc. Acetic acid (28%) . . . , . . . . . . . .90 cc. Sulphite soda. . . . . . . . . . . . . . ..30 gms. Powdered alum . . . . . . . . . . . . ..30 gms.

The azo G hard (matte) paper should be given short exposure with bright light and developed with formula recommended for azo portrait prints. If the amount of elon is doubled and the hydrochinon decreased one-half, a still softer print with more detail is obtained. For contrast plates a still softer developer may be used for the prints with the following formula:

Water . . . . . . . . . . . . . . . . . . . . ..300 cc. Hydrochinon . . . . . . . . . . . . . . . .. 4 gins. Elon . . . . . . . . . . . . . . . . . . . . . . .. 4 gms. Carbonate of soda . . . . . . . . .22 gms. Sulphite of soda . . . . . . . . . . . . ..30 gms. Potassium bromide . . . . . . . . . . . 1 gm. SOME POINTS ON RECONSTRUCTION 723

Model of the External Form

With the photographs or drawings of the sections complete, our next step is to make a model of the external form of the embryo or of a large enough part of it to insure that we have as accurate a reproduction as possible. For this the ordinary Born wax plate method is used. Wax plates of the proper thickness are cut out for the external form and piled either by the orientier guide lines or according to the photographs of the external form. It is extremely important that this external form shall be as perfect as possible, for on it all subsequent reconstructions are based, as will be seen later.

The wax plates should be piled without fusing so that they can be easily unpiled later. In making this external form of a whole embryo, it is usually best to begin piling with the larger plates from the middle of the trunk towards the top of the head, and another pile from the same region towards the tail, as when horizontal sections are used. In fact, two or three piles may be used, provided the guiding curves coincide. Or one may pile the head with the trunk, then lift off the head and turn the trunk piece upside down and pile the tail end on it. In this case the guide curves will overlap.

It is a great help to use a guide curve from a negative outline in cardboard of the external form of the embryo, made by magnifying the photograph of the external form to the proper diameter, as shown in figure 2. In horizontal series this curve should be made from a direct sagittal view of the embryo, and will of course be in the same plane as the median sagittal plane of the model. It is also important to establish the relation of the plane of the sections to this enlarged outline in order to give the proper angle to the guide curve.

The sections of different embryos, for example, may be cut at somewhat different angles to the frontal plane of the embryo, as in figure 3. So if one were piling the head end of the embryo from the middle of the trunk, it would be necessary to determine the plane of the sections in relation to the whole embryo, whether in the direction of a or b or c, etc., in order that the base-line of the cardboard guide curve may coincide with the proper plane a or b or c, etc., when it rests on the baseboard. It may also happen that the sections in a horizontal series are cut obliquely to the median sagittal plane, as is often the case. In piling the plates for the external form from such a series the median sagittal plane of the plates must be made to start at a corresponding angle to the base—board, leaning either in the right or left, as the case may be. In this way the plates are piled up with their flat surfaces parallel to a horizontal base-board.

In a similar manner, the proper angle should be used in piling plates from sections cut more or less obliquely to the other planes of the embryo. It is important that the piling be done on a board with a true surface. Overhanging parts which are likely to sag should be supported from the base-board.

Fig. 2 Method of piling with cardboard outline as guide; 1, external form piled in wax plates; 2, cardboard outline guide; 3, posts for attaching same; 4, baseboard.

Fig. 3 Outline of embryo to show different arrangements of cardboard guide a, b, c, for cross-sections, cut at different angles to the frontal plane of the embryo.

Establishing Guide Planes

Since in most series there are no guide marks, it was necessary in some manner to establish guide lines on our photographs that could be used for all subsequent reconstructions. I first thought of drilling two perpendicular holes through the entire series of plates as they stood together in the piled—up form, and then by placing each plate on its own photograph the position of the hole could be traced onto the photograph and we would thus have two orientier marks on each photograph (or drawing) that could be utilized in piling plates for future models.

A somewhat different and better method was finally adopted, which has proved very successful. After the piling of the external form was complete and satisfactory and the cardboard outline removed from the head end (for example, from a series of horizontal sections) two perpendicular posts were erected from the base—board in such a manner that a line drawn between them passed along the median plane of each plate or parallel to it (fig. 4). With a straight-edge rule a line was then drawn with a needle across the top wax plate, the straight edge resting against the edges of the two perpendicular posts, and a second line was drawn at right angles to this at a given distance from one of the perpendicular posts. The top plate was then carefully lifted off and similar lines drawn on the next and each succeeding plate in turn.

Fig. 4 Method of making guide lines: I, baseboard; .9, perpendicular posts; 3, straight edge; 4, piece at right angles to it.

Care must be taken, of course, not to disturb the position of the plates until the lines are drawn. Thus there are established on each wax plate two lines, at right angles to each other, which coincide with two planes through the model or embryo that are perpendicular to the plane of the sections. One of the principal planes either coincides with the median plane or is parallel to it, while the other is at right angles to this and at a given distance from one of the perpendicular posts. With these two principal planes established, it is possible to repile the external form or any other part of the embryo with mathematical precision, since these two planes are likewise perpendicular to the plane of the sections.

After the guide lines have been drawn on each wax plate, they must next be transferred to the photographs or drawings by super—imposing each wax plate on its photograph or drawing and marking at the ends of the guide lines. The wax plate is then lifted off and the points on the photograph connected by lines similar to those on the plates. “Then the two principal guide lines are established we have found it convenient to establish secondary guide lines by drawing other lines, 5 cms. apart, parallel to those over the entire surface of the photographs.

We have introduced lately a still better method: namely the printing of lines in red ink over the photographs from a lithographic stone. These lines form squares of 1 cm. with slightly heavier lines every 5 cms. The lines are printed to correspond with the two principal guide planes. Such lines greatly facilitate not only the plastic reconstruction work but are of especial value for graphic reconstructions. These lines will of course coincide with various planes through the embryo. The advantage in having such a number of planes will become apparent when one wishes to reconstruct small structures that are limited to a particular part of the embryo. The whole section or any part of it will thus be included in rectangles or squares of various sizes depending upon the extent of the part.

Wax Mold for Plaster of Paris Cast

With the development of these guide planes we have abandoned the usual Born wax plate method of making models, and use instead wax plate negatives into which plaster of Paris is poured. The method is as follows: Structures to be modeled are outlined on wax plates in the usual manner, and at the same time, while the plate is still in proper position under the photograph, points are pricked through into the wax with a fine needle at the corners of the rectangle in which the structure or structures outlined are included. The outline is transferred from the photograph onto the Wax plate by the use of carbon paper; tracing on the photograph with a smooth glass point. Each plate is then carefully trimmed to the rectangular shape corresponding to that outlined by the four needle points The outlined structures are then cut out, leaving holes in the plates. The plates are then piled into a perpendicular rectangular corner (fig. 5). Bridges of galvanized iron wire (ordinary iron wire will rust and discolor the cast) can be placed in position as the piling progresses to hold the various parts of the cast together. Wire, string, or cloth may be inserted into the holes of the finer structures to give strength. Gates and vents to carry plaster from one part of the model to the other and to allow air to escape were cut through suitable places as the piling progressed. It is better not to smooth the inside of the mold. Owing to the fact that the plates were trimmed into rectangles, having the four sides in the same perpendicular planes, the structures which are represented by the holes in the plates must necessarily come into the proper relation with each other. If some of these structures come to the edge of the plate at any place, they would necessarily be cut ofl by one of these planes. After the piling is completed the outside edges of the plates are fused together to prevent the plaster from leaking out. A wax plate is also fused over the side of the block if any holes come to the edge. In piling the wax plates, it is necessary to measure the height of the block of plates after the addition of each new plate in order to be sure that the plates are piled properly as regards height. It is usually necessary to scrape off a slight burr which comes along the edge of the cut.

Fig. 5 Method of piling wax mold: 1, baseboard; 2, perpendicular right angle corner; 3, glass plate; 4, wax mold; 5, vent; 6', galvanized iron wire bridge; 7, gate for plaster between parts of mold.

We have often found it advisable to build up models in rather small blocks, usually about 50 mm. in thickness, and where the models are large these blocks can be limited in other directions as well and the casts later fused together or merely fitted together as a dissectable model. Such combinations can be varied to suit special conditions.

The Plaster of Paris Cast

After the piling is completed and the edges of the plates are fused, the bottom of the pile which rests on the glass plate is made fast. Plaster of Paris is then poured into mold until it rises above the top and before it has completely hardened the excess on top is usually scraped off level with the top plate.

We used a grade of plaster known as potter’s plaster. The mass consists of about equal weights of water and plaster. The latter is sifted into the water until it just begins to show dry on the top. It is then stirred a little and poured into the mold. After setting for an hour or so, the wax is melted off in boiling water. The cast is taken out and washed in very hot water and dried in the air. Plaster of Paris is wonderful material to work with and requires but little experience to handle it with considerable facility. The plaster cast of course shows the lines of the plates just as wax models do unless considerable polishing is done. It is easier to smooth off a plaster cast than a wax model. The plaster is easily trimmed with a knife or sandpaper and the angles remaining between the edge of the plates can easily be filled in with fresh plaster painted on with a brush to any desired thickness. Corrections and additions can also be made on such plaster models by cutting off and building up with fresh plaster, using wire, string, cloth, etc., if necessary. The different structures are easily tinted with water colors (suspensions in water or ordinary commercial house paint pigments; such as, ultramarine blue, yellow ochre, burnt sienna, chrome yellow, English vermillion, etc.). A better method if one wishes to polish the model, is to mix up fresh plaster by using water colored with the pigment and to do the final smoothing with this mixture. If models are made in sections there is no very great difficulty in putting these together in the proper relation to each other. Finally the entire model can be toughened by soaking in hot paraffin until all the air is driven out by the paraflin which penetrates through the plaster. It is best to have the paraffin bath somewhere between 95 and 100°C. when the model is lifted out, in order that the surface will not be heavily coated with paraflin.

Wax Plates

The wax plates were made according to the following formula:

Bees’ wax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 parts Paraflin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 parts White lump rosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :2 parts

The ordinary 56°C. Standard Oil paraffin was used; lump rosin is much better than powdered rosin. 2000 grams poured on very hot water—surface 3 by 4 feet—gives plate 2 mm. in thickness. The hotter the wax and water the better. The air bubbles which form in the wax are driven off before the plates cool by playing a Bunsen flame over the surface.

The points which I wish to emphasize are: first, the use of photographs; second, the use of the series of guide lines which coincide with planes that are at right angles to each other and perpendicular to the plane of the sections; and third, the use of plaster of Paris. Although the preliminary steps are somewhat more complicated than those usually employed, they are nevertheless essential and in the end save both time and expense. The advantages of a plaster model are obvious to those who have worked with wax and realize the dangers of distortion and the difliculties involved in strengthening the wax and in modeling fine structures.

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Cite this page: Hill, M.A. (2024, May 18) Embryology Paper - The use of guide planes and plaster of Paris for reconstructions from serial sections. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_use_of_guide_planes_and_plaster_of_Paris_for_reconstructions_from_serial_sections

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