- Email: [email protected]
Microstructure of Tooth Surfaces as Revealed by the Electron Microscope*
772
T
he
J
ournal of the
A
m e r ic a n
lain reads a burial service for each man. Burial at sea is in complete accord with the wishes of most men of the sea. In fact, many officers and men have re quested and have received permission to be buried at sea, whether they are serv ing on land or sea at the time of death. As soon as the ship has reached the base from which she is operating, casual ties that can be transferred are moved either to a hospital ship or to a base hospital. Here, they can be taken care of more satisfactorily, since aboardship there is a great limitation of space.
D
en tal
A
ss o c ia t io n
T he ship, if she has suffered damage, is placed in fighting condition again as quickly as possible. Officers and men are transferred from the operating base to the ship to fill the positions left vacant by the fighting men who have carried on before them. It is these men who are so proudly protecting our homes, in order that our families and loved ones may live, as we have known life, in the American W ay! U.S.S. South Dakota, c/o Postmaster, New York, N. Y.
MICROSTRUCTURE OF TOOTH SURFACES AS REVEALED BY THE ELECTRON MICROSCOPE* M .S., and L a r s Ann Arbor, M ich.
A l b e r t G u s t a v R ic h a r d s , !
H E preliminary report concerns a study, begun in July 1943, of the structure of human teeth as re vealed by electron micrographs of tooth surfaces. T he report illustrates the re sults obtained when this technic is ap plied to the study of dentin. A more thorough study of dentin of various ages and locations and of the other tooth structures is under way and will be re ported when completed. T h e electron microscope has been used successfully in other fields, such as bac teriology, physics and metallurgy, and the extension of its application to the field of dentistry follows in a logical sequence. A brief description of the electron micro
T
*Investigation made possible by a grant from the Horace H. Rackham Fund. flnstru ctor in radiology, University of M ich igan, School of Dentistry. ^Associate professor of chemical engineer ing, University of Michigan, College of En gineering. Jour. A .D .A ., Vol. 31, June 1, 1944
T h o m assen ,!
Ph.D.,
scope1 is in order. This instrument can best be described by means of an analogy with the optical or light microscope. W ith both types of microscope, a beam of radiation passes through the speci men to be studied and then magnifies the emergent beam by means of a system of lenses until a highly magnified image is produced. This image m ay then be ob served visually or recorded photograph ically for future study. T h e ordinary optical microscope em ploys white light as its form of radiation, while the electron microscope uses a beam of electrons. T h e optical micro scope uses glass lenses and the R .C .A . electron microscope used in this study has magnetic lenses. T h e electron beam differs markedly from the light beam in two respects: First, the electron beam is invisible to the human e y e ; hence, a fluorescent screen is used to render visible the en
R ic h a r d s an d T h o m a s s e n — T o o t h S u r f a c e s
773
larged invisible electron im age of the specim en. Second, electrons are easily stopped by m olecules o f air, w hile light encounters little resistance from them . B ecause o f this difficulty, the electron m icroscope is constructed so th at the p ath a lo n g w hich the electron beam passes, the specim en, the fluorescent
ent the specim en is subjected to a high vacuum , it is obvious that only d eh y d rated and degassed specimens can be investigated in the electron m icroscope.
Fig. i . — T o o th m ounted in bakelite and ground to desired surface.
F ig. 2.— Piece of polystyrene bearing tooth surface impression.
A
B
F ig. 3.— a, electron m icrograph. ( X 6,000.) b, structure at center of a. (X 2 2 ,o o o .)
screen and the ph otographic plate are all kept in a h igh vacuu m . B ecause at pres-
Perhaps, in the future, new m ethods and apparatu s w ill be availab le so that
774
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
Fig. 6.— R epresentative views ( X 6 ,o o o ) of specim en shown in Figu re 5.
R
ic h a r d s a n d
T
h o m a sse n —
hydrated specimens can be viewed. Light and electron beams are alike in that both affect photographic emulsions. W hen the image of an interesting por tion o f the specimen is revealed upon the fluorescent screen in the electron m icro scope, it can be permanently recorded by merely substituting a photographic plate for the fluorescent screen. Since the electron beam is stopped so easily, even by air molecules, it is at once realized that only, very thin specimens can be used in the electron microscope. A suitable specimen should be about 1,000 Angstrom units or less in thick ness, an Angstrom unit being equal to o.ooo i micron or o.ooooi cm. In bac teria or particle-size research, the speci men itself can be supported on a thin film of collodion and placed directly in the electron microscope, but this is im possible for a calcified tooth because no sectioning method, known to us, is capa ble o f producing sections o f calcified tooth structures of the desired thickness. Therefore, an indirect method of speci men preparation must be used. T h e method of specimen preparation used in this investigation is an adapta tion of a method developed by Heidenreich and Peck2 for the study of the surface structure of metals. T h e technic herein described consists of first prepar ing the tooth surface that is to be studied. O ne method is to fracture the tooth in the plane or surface that is to be studied. Another method is to mount the tooth in a block of bakelite, using a temperature of 1350 C . and a pressure of 3,500 pounds per square inch, and then grind off the tooth and bakelite until the desired surface of the tooth is reached. (Fig. 1.) Next, this tooth sur face is carefully polished, the final polish being obtained by using four-zero metallographic polishing paper. T h e tooth is then etched in 5 per cent hydrochloric acid for about sixty seconds in order to reveal the structure of the dentin. It is entirely possible that other etching re
T
ooth
Surfaces
775
agents would reveal the dentin structure differently. An impression of the fractured or etched surface is next made in a thermo plastic resinous material, polystyrene, at a temperature of i 6 o ° C . and a pressure of 2,000 pounds per square inch. T h e tooth in its bakelite mounting is then separated from the plastic material (Fig. 2) and the tooth is discarded. T h e plas tic, bearing the impression, is placed in a silica evaporator. A quantity of silica (3 mg. for the particular equipment used in this investigation) is placed in a tung sten filament that is 6 cm. directly below the piece of plastic bearing the tooth surface impression. T h e air is exhausted from the evaporator until a vacuum of io~5 mm. of mercury is reached. A cur rent of from 19 to 20 amperes is passed through the tungsten filament, heating it to a bright incandescence. T h e silica quickly evaporates and condenses on the walls of the evaporator and on the piece of plastic bearing the tooth impression. A fter forty-five seconds, the current is turned off, since the evaporation is com plete and a thin film o f silica of proper thickness has condensed on the plastic. T h e plastic is removed from the evapo rator, and the silica film is removed m echanically from all surfaces of the plastic except the one bearing the im pression of the tooth. T h e film on this remaining surface is approximately flat on one side and faithfully follows the impression of the tooth on the other. This silica film has the same surface characteristics that the etched tooth had originally and is of proper thickness to, be used as specimen in the electron microscope. T h e film is freed from the plastic material by dissolving the plastic in ethyl bromide. From this point on, the specimen (silica film) is supported on a circular piece of 200-mesh screen of one-eighth inch diameter. O nly a small fraction of one opening in the 200mesh screen can be magnified at one time. Thus, many adjacent pictures can
776
T
he
J
ournal of the
A
m e r ic a n
be taken, all within one screen open ing. Figure 3, a is an electron micrograph ( X 6,000) of a polished and etched sur face of dentin from a mature lower right cuspid. T h e tooth surface involved in this instance was a plane running parallel to the long axis of the tooth. T h e surface was about 2 mm. away from the pulp chamber. Figure 3, b shows the structure that appears in the center of Figure 3, a, but at a magnification of X 22,000. Figure 4 presents representative views ( X 6,000) of the same specimen as Figure 3-
Figure 5, a shows a section of dentin from a fractured mature upper right cuspid at X 1,200. Figure 5, b shows the same area at X 2,800 and Figure 5, c at X 6,000. T h e pi ne in which this tooth fractured passed directly through the long axis of the tooth. O nly the coronal third of the tooth was used. Figure 6 presents representative views (X 6 ,oo o) of the same specimen as F ig ure 5. It was noted that the broad lines shown in the illustrations all had the same approximate slope and that some of the lines could be followed from one side of an opening in the 200-mesh
D
en tal
A
s s o c ia t io n
screen to the opposite side of the open ing. T h e tooth surface illustrated in Figures 5 and 6 was neither altered mechanically nor subjected to any chem ical action, so that the broad lines were not created artificially. T h e teeth used in this study were dried teeth of unknown age and origin. A fter extraction, they had been boiled in a solution of Gold Dust soap, bleached in a sodium hypochlorite solution, washed and dried. T h e fact that a particular specimen was taken from an uncertain area of the tooth surface is not of prime importance in this study because no attempt has been made to identify den tin structures, but only to illustrate the technic. These electron micrographs indicate that the technic herein described is ap plicable to the study of the microstruc ture of dentin and can easily be extended to include other tooth structures and opaque dental materials. b ib l io g r a p h y
1. B u r t o n , E. F., and K o h l , W. H .: Elec tron Microscope. New York: Reinhold, 1942. 2. H e i d e n r e i c h , R. D., and P e c k , V . G .: Fine Structure of M etallic Surfaces with Elec tron Microscope. /. Applied Physics, 14:2329, January 1943.
T
he
J
ournal of the
A
m e r ic a n
lain reads a burial service for each man. Burial at sea is in complete accord with the wishes of most men of the sea. In fact, many officers and men have re quested and have received permission to be buried at sea, whether they are serv ing on land or sea at the time of death. As soon as the ship has reached the base from which she is operating, casual ties that can be transferred are moved either to a hospital ship or to a base hospital. Here, they can be taken care of more satisfactorily, since aboardship there is a great limitation of space.
D
en tal
A
ss o c ia t io n
T he ship, if she has suffered damage, is placed in fighting condition again as quickly as possible. Officers and men are transferred from the operating base to the ship to fill the positions left vacant by the fighting men who have carried on before them. It is these men who are so proudly protecting our homes, in order that our families and loved ones may live, as we have known life, in the American W ay! U.S.S. South Dakota, c/o Postmaster, New York, N. Y.
MICROSTRUCTURE OF TOOTH SURFACES AS REVEALED BY THE ELECTRON MICROSCOPE* M .S., and L a r s Ann Arbor, M ich.
A l b e r t G u s t a v R ic h a r d s , !
H E preliminary report concerns a study, begun in July 1943, of the structure of human teeth as re vealed by electron micrographs of tooth surfaces. T he report illustrates the re sults obtained when this technic is ap plied to the study of dentin. A more thorough study of dentin of various ages and locations and of the other tooth structures is under way and will be re ported when completed. T h e electron microscope has been used successfully in other fields, such as bac teriology, physics and metallurgy, and the extension of its application to the field of dentistry follows in a logical sequence. A brief description of the electron micro
T
*Investigation made possible by a grant from the Horace H. Rackham Fund. flnstru ctor in radiology, University of M ich igan, School of Dentistry. ^Associate professor of chemical engineer ing, University of Michigan, College of En gineering. Jour. A .D .A ., Vol. 31, June 1, 1944
T h o m assen ,!
Ph.D.,
scope1 is in order. This instrument can best be described by means of an analogy with the optical or light microscope. W ith both types of microscope, a beam of radiation passes through the speci men to be studied and then magnifies the emergent beam by means of a system of lenses until a highly magnified image is produced. This image m ay then be ob served visually or recorded photograph ically for future study. T h e ordinary optical microscope em ploys white light as its form of radiation, while the electron microscope uses a beam of electrons. T h e optical micro scope uses glass lenses and the R .C .A . electron microscope used in this study has magnetic lenses. T h e electron beam differs markedly from the light beam in two respects: First, the electron beam is invisible to the human e y e ; hence, a fluorescent screen is used to render visible the en
R ic h a r d s an d T h o m a s s e n — T o o t h S u r f a c e s
773
larged invisible electron im age of the specim en. Second, electrons are easily stopped by m olecules o f air, w hile light encounters little resistance from them . B ecause o f this difficulty, the electron m icroscope is constructed so th at the p ath a lo n g w hich the electron beam passes, the specim en, the fluorescent
ent the specim en is subjected to a high vacuum , it is obvious that only d eh y d rated and degassed specimens can be investigated in the electron m icroscope.
Fig. i . — T o o th m ounted in bakelite and ground to desired surface.
F ig. 2.— Piece of polystyrene bearing tooth surface impression.
A
B
F ig. 3.— a, electron m icrograph. ( X 6,000.) b, structure at center of a. (X 2 2 ,o o o .)
screen and the ph otographic plate are all kept in a h igh vacuu m . B ecause at pres-
Perhaps, in the future, new m ethods and apparatu s w ill be availab le so that
774
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
Fig. 6.— R epresentative views ( X 6 ,o o o ) of specim en shown in Figu re 5.
R
ic h a r d s a n d
T
h o m a sse n —
hydrated specimens can be viewed. Light and electron beams are alike in that both affect photographic emulsions. W hen the image of an interesting por tion o f the specimen is revealed upon the fluorescent screen in the electron m icro scope, it can be permanently recorded by merely substituting a photographic plate for the fluorescent screen. Since the electron beam is stopped so easily, even by air molecules, it is at once realized that only, very thin specimens can be used in the electron microscope. A suitable specimen should be about 1,000 Angstrom units or less in thick ness, an Angstrom unit being equal to o.ooo i micron or o.ooooi cm. In bac teria or particle-size research, the speci men itself can be supported on a thin film of collodion and placed directly in the electron microscope, but this is im possible for a calcified tooth because no sectioning method, known to us, is capa ble o f producing sections o f calcified tooth structures of the desired thickness. Therefore, an indirect method of speci men preparation must be used. T h e method of specimen preparation used in this investigation is an adapta tion of a method developed by Heidenreich and Peck2 for the study of the surface structure of metals. T h e technic herein described consists of first prepar ing the tooth surface that is to be studied. O ne method is to fracture the tooth in the plane or surface that is to be studied. Another method is to mount the tooth in a block of bakelite, using a temperature of 1350 C . and a pressure of 3,500 pounds per square inch, and then grind off the tooth and bakelite until the desired surface of the tooth is reached. (Fig. 1.) Next, this tooth sur face is carefully polished, the final polish being obtained by using four-zero metallographic polishing paper. T h e tooth is then etched in 5 per cent hydrochloric acid for about sixty seconds in order to reveal the structure of the dentin. It is entirely possible that other etching re
T
ooth
Surfaces
775
agents would reveal the dentin structure differently. An impression of the fractured or etched surface is next made in a thermo plastic resinous material, polystyrene, at a temperature of i 6 o ° C . and a pressure of 2,000 pounds per square inch. T h e tooth in its bakelite mounting is then separated from the plastic material (Fig. 2) and the tooth is discarded. T h e plas tic, bearing the impression, is placed in a silica evaporator. A quantity of silica (3 mg. for the particular equipment used in this investigation) is placed in a tung sten filament that is 6 cm. directly below the piece of plastic bearing the tooth surface impression. T h e air is exhausted from the evaporator until a vacuum of io~5 mm. of mercury is reached. A cur rent of from 19 to 20 amperes is passed through the tungsten filament, heating it to a bright incandescence. T h e silica quickly evaporates and condenses on the walls of the evaporator and on the piece of plastic bearing the tooth impression. A fter forty-five seconds, the current is turned off, since the evaporation is com plete and a thin film o f silica of proper thickness has condensed on the plastic. T h e plastic is removed from the evapo rator, and the silica film is removed m echanically from all surfaces of the plastic except the one bearing the im pression of the tooth. T h e film on this remaining surface is approximately flat on one side and faithfully follows the impression of the tooth on the other. This silica film has the same surface characteristics that the etched tooth had originally and is of proper thickness to, be used as specimen in the electron microscope. T h e film is freed from the plastic material by dissolving the plastic in ethyl bromide. From this point on, the specimen (silica film) is supported on a circular piece of 200-mesh screen of one-eighth inch diameter. O nly a small fraction of one opening in the 200mesh screen can be magnified at one time. Thus, many adjacent pictures can
776
T
he
J
ournal of the
A
m e r ic a n
be taken, all within one screen open ing. Figure 3, a is an electron micrograph ( X 6,000) of a polished and etched sur face of dentin from a mature lower right cuspid. T h e tooth surface involved in this instance was a plane running parallel to the long axis of the tooth. T h e surface was about 2 mm. away from the pulp chamber. Figure 3, b shows the structure that appears in the center of Figure 3, a, but at a magnification of X 22,000. Figure 4 presents representative views ( X 6,000) of the same specimen as Figure 3-
Figure 5, a shows a section of dentin from a fractured mature upper right cuspid at X 1,200. Figure 5, b shows the same area at X 2,800 and Figure 5, c at X 6,000. T h e pi ne in which this tooth fractured passed directly through the long axis of the tooth. O nly the coronal third of the tooth was used. Figure 6 presents representative views (X 6 ,oo o) of the same specimen as F ig ure 5. It was noted that the broad lines shown in the illustrations all had the same approximate slope and that some of the lines could be followed from one side of an opening in the 200-mesh
D
en tal
A
s s o c ia t io n
screen to the opposite side of the open ing. T h e tooth surface illustrated in Figures 5 and 6 was neither altered mechanically nor subjected to any chem ical action, so that the broad lines were not created artificially. T h e teeth used in this study were dried teeth of unknown age and origin. A fter extraction, they had been boiled in a solution of Gold Dust soap, bleached in a sodium hypochlorite solution, washed and dried. T h e fact that a particular specimen was taken from an uncertain area of the tooth surface is not of prime importance in this study because no attempt has been made to identify den tin structures, but only to illustrate the technic. These electron micrographs indicate that the technic herein described is ap plicable to the study of the microstruc ture of dentin and can easily be extended to include other tooth structures and opaque dental materials. b ib l io g r a p h y
1. B u r t o n , E. F., and K o h l , W. H .: Elec tron Microscope. New York: Reinhold, 1942. 2. H e i d e n r e i c h , R. D., and P e c k , V . G .: Fine Structure of M etallic Surfaces with Elec tron Microscope. /. Applied Physics, 14:2329, January 1943.