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TRANSCRIPT
HIGH-RESOLUTION AUTORADIOGRAPHY
WITH 33P
APPROVED:
Graduate Committee:
Minor/jProf essor
Ji, (2t Committee Membe
Committee Member
Cor tee Member
\;A nA MA? f the Depart
(4*ACt
•\ rC romittee Member
Directajf^if the Department of Biology
Dean o£ the Graduate School
HIGH-RESOLUTION AUTORADIOGRAPHY
WITH 33P
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
4
Paul R&. Holmgren, B. S., M. A. A. l |
Denton, Texas August, 1969
TABLE OP CONTENTS
Page
LIST OF TABLES iv
LIST OF ILLUSTRATIONS v
Chapter
I. INTRODUCTION '1
II. METHODS AND MATERIALS 5
Liquid Scintillation Studies Emulsion Sensitization Preliminary Study Autoradiographic Studies
III. RESULTS * . . * . . . . . 12
Liquid Scintillation Counting Preliminary Sensitization Studies Autoradiographic Studies
IV. DISCUSSION . . . . . . . 31
Liquid Scintillation Studies Autoradiographic Studies
V. SUMMARY 44
BIBLIOGRAPHY 45
LIST OF TABLES
Table Page
I* Uptake of 33P by Skin, Liver, Muscle and Kidney Tissue . . . . . . . . . . . i.15
II. Loss of Radiophosphorus Due to Preparation for Electron Microscopy . . . . . . . . . lb
III. Distribution of Radiophosphorus in Electron Microscope Preparative Solutions 15
LIST OF ILLUSTRATIONS
Figure Page
1. An Electron Micx-ograph of Monolayer of Silver Halide Crystals from Kodak NTE Emulsion . . . . . . . . . . . . 9
2. Emulsion Covered Grid from Group A Exposed to Strobe Light Flash Only . . . . . . . . 16
J. Emulsion Covered Grid from Group B Exposed to 33P Only 17
4. Emulsion Covered Grid from Group C Exposed to Both Strobe Flash and Radioactivity . . 18
5. Portion of a Hepatocyte from a Control Mouse 19
6. Autoradiogram of a Hepatocyte after Two Hours Injection with 33P 20
7. Hepatocyte from a Mouse Sacrificed Two Hours after Injection with 33P . . . . . . 2 1
8. Hepatocyte from a Mouse Sacrificed Four Hours after Injection with 33P .22
9. Hepatocyte from a Mouse Sacrificed Twelve Hours after Injection with 33P . . . . . . 2J
10. Autoradiogram of Muscle Tissue from a Mouse Which Received No Radiophosphorus . . . . 24
11. Muscle Tissue from a Mouse Sacrificed Two Hours after Injection with 33P 25
12. Autoradiogram of Proximal Renal Tubule Cells from a Mouse that Received No Radiophosphorus . . . . . . 26
Figure Page
15. Renal Proximal Tubule Cells from a Mouse Sacrificed after Two Hours of 33P Uptake 27
14. Autoradiograms of Control Skin Tissue. Germinal Epithelial Cell . . . . . . . . . 2 8
15- Autoradiogram of Skin Germinal Epithelium from a Mouse Sacrificed Two Hours after Injection with 33P . . . . . . . . . 2 9
vi
CHAPTER I
INTRODUCTION
Phosphorus is an essential constituent of every living
cell and its compounds have more functions than any other
single nutrient (4). Detecting the precise intracellular
location of specific phosphorus compounds can, therefore,
be a very useful tool to the cell biologist. Intracellular
autoradiographic studies using radiophosphorus are, never-
theless, rare, because of two basic problems. First, the
radiophosphorus compounds normally used, such as sodium or f
potassium radiophosphate, are very soluble in aqueous solu-
tions^ and thus are subject to considerable diffusion in
excised tissues. The aqueous solutions normally used to fix
tissues for electron microscope investigations add to this
problem. Second, the radiophosphorus normally used by cell
biologists, 32P, emits beta particles whose energy level is
too high to permit determination of the intracellular local-
ization of labeled phosphorus compounds by the standard
autoradiographic techniques. The emitted' beta particle,
with energies up to 1.71 MeV, passes through the overlying
small-grained emulsions without imparting sufficient energy
to expose the silver halide crystals (8). Crystals large
enough to become exposed in this manner are too large for
high-resolution autoradiography (2). Also, the long range
of the emitted beta particle, combined with its tortuous
path (7), will cause exposure too far away from the source
to give adequate resolution (5). The larger-grained emulsions,
which are necessary when using high energy sources, increase
the possibility of distant exposure and lower the resolution
accordingly (2). All of these problems combine to malce high-
resolution autoradiographic studies of S2P compounds imprac-
tical.
Another radioisotope of phosphorus,^33P, has only re-
cently become available commercially. S3P is strictly a beta
emitter, with a maximum energy of 0.25 MeV and a half-life
of 24.4 days (5). These characteristics make 33P a very use-
32
ful radioisotope for the cell biologist. For example, P and
s 3P can be combined in double-labeling experiments. Experi-32
ments of longer duration than those employing P are made
possible by 'the greater half-life of 33P. The lower energy
of S3P, as compared to 32P, gives higher photographic sensi-
tivity, better autoradiographic resolution^ smaller brems-
strahlung doses, and lower recoil energy (10). 33P is pro-
duced by a (n, p) reaction on sulfur enriched in 33S (10).
S3P decays to 33S according to the reaction 33p—>-33g + p
+ v (9)-
Possibly the first person to employ 33P in autoradiography
was Mayr, who used autoradiography to determine small amounts
of 33P present in samples of 32P (6). Apelgot and Latarjet
compared the "suicide" rate of bacteria marked with 32P and
33P (1). They found that the lethal effect of S3P decay was
due not to the recoil energy applied to the newly-formed sul-
fur atom, but rather to the jLn situ appearance of the sulfur
atom.
The purpose of this study is to provide groundwork for
future autoradiographic investigations using 33P as a tracer.
Although 33P is a more desirable isotope for autoradiographic
experiments than 32P, its energy level is still too high to
expose the small-grained emulsions necessary for high-resolu-
tion autoradiography* Therefore, this study presents a
feasible method for sensitizing the small-grained emulsion,
thereby making high-resolution autoradiography possible with
33P.
CHAPTER BIBLIOGRAPHY
1. Apelgot, Sonia, and Raymond Latarjet, "Comparison of -•Suicides' of Bacteria Marked by Radioactive Phos-phorus 32 and 33/" International Journal of Radi-ation Biology, X (Feb., 1966), 165-175-
2. Bachmann, L., and M. M. Salpeter, "Autoradiography with Electron Microscope. A Quantitative Evaluation," Laboratory Investigation, XIV (June, 1965)/ 10^1-1053-
3. Caro, L. G. and M. Schnos, "Tritium and Phosphorus-32 in High Resolution Autoradiography," Science, CXLIX (July, 1965), 60-62.
4. Gilbert, Frank A., Mineral Nutrition and the Balance of Life, Norman, Oklahoma, University of Oklahoma Press, 1957-
5. Mace, Robert C., personal communications, New England Nuclear Corporation, Boston, Massachusetts, 1969.
6. Mayr, J., "The Use of Nuclear Emulsions to Determine Small Amounts of p 3 3 Present in Samples of p^2," Experientia, XI (April, 1955)' H -
7. Oldenberg, Otto and Wendell G. Holladay, Introduction to Atomic and Nuclear Physics, New York, McGraw-Hill Book Company, 1967-
8. Rogers, Andrew W., Techniques of Autoradiography, New York, Elsevier Publishing Company, 1967-
9- Sheline, Raymond K., Richard B. Holtzman, and Chang-Yun Fan, "The Nuclide p33 and the p32 Spectrum," Physical Review, XXCIII (September, 1951), 919-923.
10. Westermark, E. G. T., I. G. A. Fogelstrom-Fineman and S. R. Forberg, An Approach to the Production of Phosphorus-33 in Millicurle Quantities in Radioiso-topes _in Scientific Research, Volume I, edited by R. C. Exterrnann, New York, Pergamon Press, 1958*
CHAPTER II
MATERIALS AND METHODS
Disodium'phosphate (Na2H3SP04) obtained from Tracerlab
(waltham, Massachusetts) was injected subcutaneously into
DBA/1 J mice, so that each received twenty microcuries per
gram of body weight. Mice were sacrificed by cervical dis-
location at two hours, four hours, and twelve hours. Samples
of skin, liver, kidney cortex, and striated muscle, weighing
approximately twenty milligrams, were removed from each
mouse at the time of sacrifice, making a total of twelve f
tissue samples. Each of these samples was then cut into four
pieces and distributed into four groups. Consequently, each
group contained tissue from the original twelve samples.
The twelve samples of one group (i) were weighed, frozen,
and later hydrolyzed for liquid scintillation count analysis.
The twelve samples of a second group (il) were weighed and
fixed in glu.taraldehyde for forty-five minutes and post-fixed
in osmium for two hours at four degrees centigrade. Both
fixatives were buffered with sodium caco'dylate at pH f .2.
These tissue samples were then dehydrated in a graded series
of alcohols for forty-five minutes, placed in propylene
oxide for ten minutes, and hydrolyzed for liquid scintillation
counting. The samples of a third group (ill) were placed
directly into osmium buffered with an isotonic solution of
phosphate buffers at pH J.2, after the method of Millonig (3).
After being fixed in this solution for two hours and forty-
five minutes at four degrees centigrade, they were dehydrated
and hydrolyzed in a manner similar to Group II. The tissues
of a fourth group (iv) were treated similar to Group III,
but were embedded in Epon (2) for autoradiographic studies.
A fifth group (v), comprised of tissues from a control mouse
which had received no radiophosphorus, were treated similar
to those of Group IV.
Liquid Scintillation Study
The tissue samples of Groups I, II, and III were hydro-
lyzed in 20 per cent sulfuric acid at eighty degrees centi-
grade and diluted to two milliliters with aqueous sodium'
hydroxide to raise the pH to approximately 7-0- A 0.2-milli-
liter aliquot of each sample was placed in ten milliliters
of scintillation cocktail. The cocktail consisted of six
grams of Beckman PPO (2 5-^iphenyloxazole) and 0.06 grams of
Beckman POPOP (l 4-D [2-(5-phenyloxazolyl)]) in one liter of
distilled toluene. To this cocktail, 200 milliliters of
Beckman Bio-Solve BBS-5 was added to make the final, cocktail
a solvent for the 0-2-milliliter aliquots of aqueous samples.
The samples were counted in a Beckman LS-100 Liquid Scin-
tillation Counter equipped with a direct data read-tout
module and an auto-quench mode. Each sample was counted
twice for two minutes each and read in counts per minute
using a fixed-window isoset calibrated for 3H, and 32P.
Emulsion-Sensitization Preliminary Study
A Sunset strobe lamp* normally employed in photography,
was used to sensitize the emulsion immediately before expo-
sure to 3,JP. The flash of light, lasting approximately
1/1,500 of a second, was passed through a Bessler Model 45
MCRX enlarger equipped with a 105-millimeter lens. For this
procedure, the enlarger lamp was removed and replaced with
the strobe lamp, which was positioned 56 centimeters above
the enlarger lens. The amount of light transmitted with
the lens eight inches from the emulsion-covered grid and' the
diameter of its iris diaphragm set at 0-7 centimeters sensi-
tized the emulsion without producing background density.
For the preliminary sensitization study, collodion-
covered grids coated with emulsion were placed, emulsion side
up, over radioactive areas on filter paper. These areas of
8
radioactivity were prepared by placing drops of concentrated
Na H s sPO (approximately one microcurie per milliliter) on
pieces of filter paper and allowing them to dry. The emul-
sion-covered grids were exposed for a period of thirty
minutes under the same conditions as described for the auto-
radiographic study.
Autoradiographic Study
Tissue from Groups IV and V were sectioned at approximately
ninety millimicrons with a Sorvcill MT-2 Ultrarnicrotome
equipped with a diamond knife. Sections,,placed on collodion-
covered stainless steel grids, were stained with uranyl ace-
tate for five minutes and with lead tartrate (4) for five
minutes. The sections were then covered with a layer of
evaporated carbon fifty angstroms thick.
Kodak NT33 emulsion was prepared for grid coating by
diluting it one to ten with glass-distilled water at fifty
degrees centigrade, centrifuging the diluted emulsion at
5,000 RPM for one minute, discarding the pellet, and spinning
the supernatant at 8,000 RPM for five minutes. It was
necessary to heat the rotor in a water bath to sixty degrees
centigrade prior to each centrifugation in order to keep
the emulsion at fifty degrees. After centrifugation at
8,000 RPM, the emulsion, which remained at the bottom of the
centrifuge tube, was used to coat the grids. A uniform mono-
layer of silver halide crystals was obtained by the drop
method of Salpeter and Bachman (5 ) or the loop method of
Carl and van Tubergen (1). Figure 1 shows an electron micro-
graph of a typical monolayer obtained by the loop method.
The grids with the emulsion-covered tissues were exposed in
small petri dishes covered with aluminum foil and enclosed *
in a dry/ light-tight,. air-tight box covered with several
layers of heavy-duty aluminum foil. The box was surrounded
by lead (quarter-inch thick) for the duration of exposure,
to minimize background exposure. All grids examined in the
: x L . — ' ** ^
J , )<
X " V
f I p.' k);:"
f r , J i
' / ' '
• /
> > .. *
\ '?
J
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-,A
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if
-"'"V '/ i k
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• y-
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• < J'* <
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Fig. 1—An Electron Micro.graph of a Monolayer of Silver Halide Crystals from Kodak NTE Emulsion. 51/000 X.
10
electron microscope, with the exception of the one shown in
Figure 1, were developed in Kodak Delctol for two minutes and
fixed in Kodak Rapid Fix for two minutes.
CHAPTER BIBLIOGRAPHY
1. Caro, L. G. and R. P. vail Tubergen, "High-Resolution Autoradiography. I. Methods," Journal of Cell Biology, XV (March, 1962), 173-188. ~ ~
2. Luft, J. H., "Improvements in Epoxy Resin Embedding Methods," Journal of Biophysical and Biochemical Cytology, IX (October, 19^l), 409-414.
3. Millonig, G., "Further Observations on a Phosphate Buffer for Osmium Solutions in Fixation," Fifth International Congress for Electron Microscopy, Vol. II, edited by Sydney S. Briese, New York, Aca-demic Press, 1962.
4. , "A Modified Procedure for Lead Staining of Thin Sections," Journal of Biophysical and Bio-chemical Cytology, XI (November, 1961), 756-739.
5. Salpeter, M. M. and L. Bachmann, "Autoradiography with the Electron Microscope. A Procedure for Improving Resolution, Sensitivity and Contrast," Journal of Cell Biology, XXII (July, 1964), 469-477. ~~
11
CHAPTER III
RESULTS
Liquid Scintillation Study
All of the tissues from Groups I, IX# and III of this
study were analyzed quantitatively for 33P uptake by liquid
scintillation counting. Fresh samples from each of the four
tissue types found in Group I were analyzed to determine the
relative uptake by each tissue at two hours, four hour s; and
twelve hours after injection with 33P. These results are
corrected for time of sacrifice, and presented in Table I.
The results of this study show that, of the four tissues
studied, most of the radiophosphorus is incorporated by the
liver within two hours. At this time a count of 152,190
counts per minute per milligram (wet weight) of fresh tissue
(CPM/mg) is recorded in the liver. The amount of radiophos-
phorus in the liver is decreased to 18,^00 CPM/mg at four
hours and to 6,9^0 CPM/mg at twelve hours.
In the kidney, the highest recorded level of 33P activity,
21 ,120 CPM/mg, is found at two hours. This amount is reduced
to 6 ,220 CPM/mg at twelve hours. The CPM/mg recorded in the
muscle decreases from K, 560 at two hours to 5,060 at twelve
15
hours. Of the four tissues studied, the lowest radiophos-
phorus activity is found in the skin.
TABLE I
UPTAKE OF 33P BY SKIN, LIVER, MUSCLE, AND KIDNEY TISSUE
— - •— "I
Tissue Time of Sacrifice ±
CPM/mg
Skin 2 hours 2,000 4 hours 2,000 12 hours 1,600
Liver 2 hours 152,190 4 hours 18,400 12 hours 6,9^0
Muscle 2 hours 4,560 4 hours 3/7^0 12 hours 3,060
Kidney 2 hours 21,120 4 hours 12,480 12 hours 6,220
% Counts per minute per milligram wet weight»
Tissues treated with routine electron-microscope prepar-
ative solutions were also analyzed and their 33P content
compared with the untreated fresh tissue of Group I. A com-
parison between Groups I and II was made to determine how
much leaching of 33P occurred during a typical procedure
using glutaraldehyde and osmium fixatives in a non-phosphate
14
buffer such as cocodylate. A similar comparison was made
between Groups I and III to determine how much leaching
occurred during a procedure using osmium in a phosphate buf-
fer system. Table II shows how much 33P remains in each
TABLE II
LOSS OF RADIOPHOSPHORUS DUE TO PREPARATION FOR ELECTRON MICROSCOPY
Tissue Glutaraldehyde-osmium fix-ation in cacodylate buffer
Osmium fixation in phosphate buffer
CPM/mg* Per cent of radiophosphorus remaining after
treatment
CPM/mg26 Per cent of radiophosphorus remaining after
treatment
Skin: 2 hr. 4 hr.
12 hr.
360 660 920
18 .0 33.0 5 7 . 5
f
810 1 ,550 1,380
4 0 . 5 7 7 . 5 8 6 . 3
Liver: 2 hr. 4 hr.
12 hr.
15/460 11,120
4 ,680
8 . 8 6o .4 67 .4
28 ,090 14,570
4 ,900
1 8 . 4 7 9 . 2 7 0 . 6
Muscle: 2 hr. 4 hr.
12 hr.
5 ,300 1 ,060
720
7 2 . 3 2 8 . 3 23 -5
3 ,780 2 ,070 1 ,980
80 .7 5 5 . 3 6 4 . 7
Kidney: 2 hr. 4 hr.
12 hr.
18,620 7 ,300 3 ,840
8 8 . 2 58 .4 6 1 . 7
17,880 9 ,270 5 ,230
84 .7 7 4 . 2 84 .0
^Counts per minute per milligram of tissue after treat-ment with electron microscope preparative solutions (see Table l)
15
tissue after these two treatments. In every tissue large
amounts of 33P are lost to the various electron-microscope
preparative solutions. The amount lost in the phosphate-
buffered osmium fixative procedure is less than the amount
lost in the non-phosphate fixative procedure. This indicates
that less leaching and possibly less diffusion of 33P are
occurring in the phosphate-buffered system.
The electron-microscope preparative solutions used in
the above experiments were retained for liquid scintillation
analysis. Table III shows that most of the 33P lost, from
the tissue is found in the initial fixative. With cacodylate
buffer, glutaraldehyde and its wash solutions retain an
average of 62.6 per cent of the total amount of the 33P lost
TABLE III
DISTRIBUTION OP RADIOPHOSPHORUS IN ELECTRON MICROSCOPE PREPARATIVE SOLUTIONS
Per Cent of Total Radiophosphorus Lost to Solutions
Glutaraldehyde Osmium Alcohol Propylene Oxide
Cacodylate Buffered Fixatives
62.6 25.7 7-7 h.i
Phosphate Buffered Fixative
— 89.8 9.0 1.2
16 r
from the tissues studied, Cacodylate-buffered osmium fixa-
tives, along with their wash solutions/ retain an average of
25«T P e r cent of the total amount of the S 3P lost from the
tissues studied. Together these total 88.3 per cent/ almost
the same as the 89-8 per cent lost in the osmium fixative
buffered with phosphate and its wash solutions. Thus, inmost
of the radiophosphorus removed by the electron-microscope
preparative solutions is taken up by the fixatives, compara-
tively little by the alcohols, and very little by the propy-
lene oxide solvent for Epon.
Fig. 2—Emulsion-covered Grid from Group A Exposed to Strobe Light Plash Only.
17
Emulsion Sensitization Preliminary Study /
Even though the radiophosphorus used in this study has
a far lower energy level than 32Pt it apparently is still too
high to expose the silver halide crystals. Therefore, it was
necessary to find ways to increase the sensitivity of the
emulsion. All but one of the numerous methods tried increased
the background density to a point unsatisfactory for this
study. Light from a strobe lamp within a narrow range of
intensity sensitized the.grains for further exposure without
causing any observable increase in background density.
V «
' • X
•j * v •'
V /
if
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Fig. 3—Emulsion-covered Grid from Group B Exposed to s aP Only*
18
In these studies emulsion-covered grids without tissues
were used. Group A (Figure 2) v/as exposed to the strobe
flash onlyj Group B (Figure jj), to S3P only; and Group C
(Figure 4), to the strobe light prior to S3P exposure. The
amount of strobe light exposure on Groups A and C was identi-
cal, as well as could be determined. Similarly, the amount
of radiation exposure on Groups B and C was identical, as
well as could be determined. Figure 4 shows that the number
of developable grains is'far greater after combined exposure
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19
than the sum of each of its parts.
Autoradiographic Study
Since diffusion occurred in tissues examined by auto-
radiography, it was necessary to examine several grids in
order to draw the following conclusion about the _in vivo
location of SSP. The figures included in this paper are
intended to be typical examples of these observations.
Liver tissue absorbed more 33P than any other tissue
studied/ and most of it appeared to be in the soluble
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Fig. 5—Portion of a Hepatocyte from a Control Mouse (31,000 x).
20
fraction of the cell. This.agrees with Scholes (6), who
found that most of the 3 2P activity in liver after two hours
is in the acid-soluble fraction of the cell. Figure 5 shows
a portion of a hepatocyte from a control mouse ('Group v )
which received no 33P. Figures 6 and j are electron micro-
scope autoradiograms of hepatocytes from mice (Group IV)
sacrificed two hours after injection of S3P. These auto-
radiograms indicate that most of the radiophosphorus is in
the soluble fraction, but some is found in the mitochondrial
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Fig. 6—Autoradiogram of a Hepatocyte Two Hours after
Injection of the Animal with 33P (75/000 x) *
21
r
and nuclear fraction as well. Four hours after injection,
more radioactivity is concentrated in mitochondria and nuclei
of the hepatocyte than is seen cit two hours. A higher con-
centration of S3P in the mitochondria at four hours than at
two hours is indicated in Figure 8. By twelve hours after
injection the overall radiophosphorus activity in the liver
cell is much reduced/ but appears to be more highly localized
in the mitochondria than anywhere else* This is indicated by
Figure 9.
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Fig. 7—Hepatocyte from a Mouse Sacrificed Two Hours after Injection with 33P (20*000 x).
22
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Fig. 8.—^Hepatocyte from a Mouse Sacrificed Four Hours after Injection with 33p (31,000 x).
Some inconsistencies in the shape of the developed
silver grains and overall density over liver tissue were
found when autoradiograms of liver tissue were compared
to those of other tissues studied. The developed grains
shown in Figure 6r for example (note higher magnification),
are smaller and more spherical than usual. Normally,
the developed grain appears longer and sometimes twisted,
as shown in Figures 2 or 11. This suggests some kind
of negative chemography resulting from diffusion of
25
• • A ^ , . ,
a V'-•• >' v.- •; ?>:• - 5 • f,r% t:-w **.-• 1 ', /*</; V. .;/> • ?; '•••: •• .,\r -y,, • . . . ,.;} •. ^
m* , ... : .. I.* . ...' - A - a* >
t
| .., •: .•••" •?. ,w'.' ;•<. ••• W.^?;i^ f-'* -S' <•,*'•• 7 ; >
i & : > M 'V \ A ,.V; •; /;$. : : r ••/'. / ,*• '* » *A7 •-. .t'A -
a ^ S ' j-7, v • ••%:•;• •" xs • , -v *; ;• S . \ "• .V' , *>. » *-» s''' *v. . "3, •>• '7 v .<'•>( . »• •. ;' * -.»* v» . *. ' ."• " * v
>••*>:* 7 • • • vw-:-,;. - *.'* -x,-v: . v.-. A * X, •• - V, -r- i<; •- .. 7* AIA'-A' f K A ; , ; A A A * 7 A C VAA<'V::AA-- 1 % 4 ' A ^ A ' A A J /••5' . K" • ." r V'X: i*VA A' /«" ) *•>•; ? ?V.i '•:'£? />Kt'4,f-A | -*!i> ,"•.••>'/' v : . 3 " . . - : - fi-.-'i*
... -:.^S • l X X ' . : X \ X . K : r ' ; • . • • ? ^ ••;]! •
. . y X - X X ^ i X X • ^
• v ' f v X x s X X ' '
' • '' .'••% • lh' "hi. .- • ••.'• 'i,•••>-.. -V/-- •,-'4 '-4 . ' '• . ' "«u vA; v -: <-:. ?$&-\ <• • \ \ ••^X A •rf ' "v. C x*"&
4- >" A :* 1.-A «.*• • <«» • /./; 'J »,»: >v . ' *. > . $> *
•' -* , - # ® - V.,# . _ *- * *& A>< v' f%k ' .;. 4X1' f.J,, ' /v X ^ ^
Fig* 9.—Hepatocyte from a Mouse Sacrificed Twelve Hours after Injection with 33p (51/000 x).
substances from the liver to the emulsion. This was not
observed to the same degree in studies of the other three
tissues. In addition, there was less overall density
observed in liver autoradiograms than was expected from '
the liquid scintillation results. This may also be
related to the negative chemography described above. It
appears that something remaining in liver tissue after
fixation interferes with the developability of the exposed
silver halide crystals.
2h
^ - s S S ^ s S K f t
K ,- '-/';:-" v '^.rs^Jv-^h o:': <;V^ ' ••'••;:':': .. i'':; -; .S'•' '
V>VS.SKSS- V|K:S'y";, ;"V':'S';,;:V'v ::- y?-:^ V |
i^} v. »*-",."- »• ?KS
k "::;;.
SV V V*J • V- . \ ' ,;. "• , - .-S/. . . . VV/;;y/;$
KS;V\ 'v ' -;V.S7-KC\JX'S'S;> V^VV^cv; .-• • ;- • . f; v ': 4 •/• •. v *. ' '• • " • - " '. ..." . vv* v.\vm '*.* •.'") :A,~* >" % . - ">* U- , >'?. '""" - r J t "vvV :«"..• 7 • 7- • "'v *V;*V'AV«Y>:A?
•;;:::>:v-i'-;-
' ' ' t-'Cfy^W'
*v * "svb > KSKrv :-V-S fv KV vV , iv;-: . „
;v:"V';V- \"„, . ' * '-• 'V V:* " ' ' '-• 'J*'' S.-'" -X K • • •« ,
":-Vip.;/ 7 •;/:•' : ' V > .V % -'* ;• VV | >'J[ f ;.ir.;vs v-
(
Fig. 10.-~Autoradiograrn of Muscle Tissue from a Mouse which Received no Radiophosphorus (31,000 X).
Autoradiographic analysis of striated muscle tissue
indicates that, after two hours of uptake,, most of the radio-
phosphorus is located in the highly vacuolated sarcolemma.
Figure 10 is an electron microscope autoradiogram of muscle
tissue from a control group (v). Figure 11 is an autoradio-
gram of muscle tissue from a mouse from Group IV saqrificed
two hours after 33P injection.
Electron microscope examination of kidney autoradio-
grams indicates that the injected radiophosphorus is
25
m-- -wmwir {^i-kr%i:v;-sis:®:; x
&{&-&*';'y':S -'•'*<? • ' ' f e r - :•'?'?. f " / T SI '
4.- :rS;0:; '•• r~-'*<'\;- V'. - '" • 'f '''""vif ...' •_ • .'.v.... ••• ::.'--v • v:.* .--r- .>5-: - ; *!Vv'.":V,-" •? / ,. \\; >*!,- A/ ; .:> „. ,„ • •' v.---1 j, \;s„; .f "*. / -
:v. r- ^v"- ,"<v V -1 .M'~VUpAffl"P::fr-•<•, fe'/v.- •••","C-;'?
^ <" * '/••" ' -v';. \ y-'f- --> *' I . •'•>.•> : A - *\ - f'j". '* - — - :>v. f I '$*<* - fc
: - . : V - - V ' / . . • -..wV;:>::\vS. (c-v.{„ * ; • , • .^ip,: v-:
h. •••, ;-K:r;v -.v.• •/. •• : •' •->-•• ;:iv •'•••'•"••••< 'vvyOAv v };•/: fv.' ' "r V;.- • , > : :.f- .. - - >• Y YYY 'Y>Y>':''
^ • * '•* ''v•v r", ~ .f'' * *v»-rVv ** -t ^ ii' .'r
Fig. 11.—Muscle Tissue from a Mouse Sacrificed Two Hours after Injection of 33P (31/000 X).
involved in a great amount of biological activity. Figures
12 and 1J are autoradiograms of areas within the proximal
renal tubule cells of a control mouse and a S3P injected
mouse, respectively. Figure 12 indicates that 33P is found
in the intercellular spaces of the proximal tubule cell.
In addition, there appears to be a rapid uptake of S3P by
the mitochondria of the proximal tubule cell. Further
electron microscope examination indicates that the mito-
chondria of these cells absorbs 33P faster than those of
26
;' •# . ;• . ; *1 1,VaV- .• >*V : , *-: Vr ' * -,.C" r*«.; {*" jr-1* ^ f"; c ' . " »v f: -v"V ,-/-/> 1 /: ^ -v ' •% . ••'<; < ' , •,•>'- •••'• aj-? ..< t'-''4
• •'•'• '• ' .*» '. ; <-* V ';-H4
.. ; 1; V- ".
, - ' " i •' • 'i i, % *.• * v i - •> f 1 14 , /,
- ' • /, ,/ -1,; A'-"% /[„ t -f j vr , V V ,•* % >,V' % 1J ' ' ' ' 1, e M" • *'i
: X ; ' '••:•••••• '•••i'V' f \ r- ' ^\f.y * - ' -/ , " - • '•'• ^ , i)'if • , ' » j' -s « ; ' f '-C- ;
C •, : . \ * i ,s ' . h - - > V\% ^ it . . > V,*' A\. ' ^
^ ^ r",r -,: • n : " •
v -*\ '• - ". \
0 " . - . r ' « • • ' \ , ' , s * .
^ -1i: >#;', ' 'V ' &
s , ' i ,» , v"
• < ; ' < i e ~ <
• y ;
,, "V > \ i ' ' ' '
s y
V ' :1" Lr
,, *
:1" Lr ' :*V l ' £ " -• .
tJ-' * J \ ' - y ;
v /v • , ' l % f
tr k •
h
/ ' '- , Xy: '*W~-
' tt I* /»rM v, , / v\ t- i. r. y' >
«: • -. • - ' i :f •! -A\ \\ - • .f n', V ' , •-'' .' ' , / -f 4-1 *• r % - ', ''{V *1 - : •"-SV '
,irt
•' V f •' •• • - V - ' / • { - • " } %«••.'': W'Vi. ;* "•'... v;
^••' >• ^ y H ;>i-v
j ' . ; ¥ •: ^ ^ 7/1; • -r $ >1
Fig. 12.—Autoradiogram of Proximal Renal Tubule Cells from a Mouse that Received no Radiophosphorus (31,000 x).
the other tissues studied. Observations of the glomerulus
(not shown) indicate that the soluble phosphate molecule
readily passes across the basal membrane into the lumen of
the proximal tubule and is partially reabsorbed by the prox-
imal tubule cell. This passage of phosphate through the
proximal tubule cell to the bloodstream is indicated by
Figure 1J. The round, slightly dense spots seen in the
mitochondria of Figures 12 and 13 are probably calcium de-
posits and not reduced silver.
it . 1 4 '* - - ' ' v'' *'< * " ' 'V ^ * • \ . !v . *
27
»" , „. "- '' -* *.* - '• ' ,/<>- ;' " * . t Xi <V'^ f-' ^ r " \ 1 / v' . ' *- —T,^'Av - ~V7 / :•/-•> %} \ ~;1 v-*-, -4
'c "V"- - , , f \ ;• - r / r . M
* * * "'% -' ' M \ , r • =:v ;: :r>
- ••- b' v,• <:w,rc. '--v;w - : •" 'V„-v-:r . ;i •;> ^r. <• v , -> V " •,: v' . ?U ** .--'---*''4
/X- ". • / ••: , ... . . ..... K-"''-'•': - , v _ : ; £ r : - ' ' • * • " ' • * >'• : ' - . - > - • : " v - : - = ' ? ' • - > - ' " > ' y
->''• it . * '- -, . ' .. ' •* "- •-, **r - * - V "' , i, ' -
•K „ „ i * 5;'*V- " \." V'*":' vV-V:V*;A-i;;^-/v -* - % -- - ,v *
-j •- - • V",'- "!'"--.;-;r •" %• ,.- v •-* - ; T, ^ ~r .-
, r- *- ~i ' • *-» * * ^ - *r . -.. ..I :':r,;-'-r>. *.J
f - .• -::•••••. '-. :->••' . : •? - .- .. -.» •', • •' • • **. '?t '"--. ' • ' ' ?tiJ ' , -' .-7 i-y • .
.>*-» - . ^ ••-• V ri( •"-,•-;•.':• >
-V ^ . •*' I - *• -- -: - •*:
r- . y • - -r. • -•' >*
-. *-vf .
• vx" * * - - ''•»• -JU-i • -" - ' *
Fig. 15.—Renal Proximal Tubule Cells from a Mouse Sacrificed after Two Hours of SSP Uptake (51,000 X).
Autoradiographic studies, as well as liquid scintil-
lation studies, of the skin indicate a low amount of 33p
uptake at two hours. The examination of several grids was
necessary to conclude that exposure was due to radiophos-
phorus and not background density. This tissue exhibits
the minimum level of radiophosphorus activity necessary to
expose the emulsion using this technique.• This slight ex-
posure becomes.evident when comparing Figure 15 to its
control Figure 15* . Most of the activity appeared to be in
28
"• ••• - %V;.. ,-V • , ,r- ••" .;•> • .. '7: • .>,• V: <
% ; . A
• * > • ' ^
Ji. V
sv . * y>. * *h
..V* .•> ';r - **'> , 7-V-; 1
^ > /fA £
& ft 'v".-• Vv-V "•/ *w-|: " •-'••;• *:*j v.-/ .•; ••; ••.. W •' ^
V - - v . ; - 1 .. * v/-, '3. '• ?V*' '. \ ' '-is ' *
-v v- - rv „ .: V- Tiv ,'jH1 - 45W fcvi *"' ::' K '> V ' k ; ' : L i M
J? l'^,. : • • '"""" ;.v„ .• .. •, . "f ;.- /• - , . *• t xHit-/""* r * • -• >- \ • t . " - . , i A,, , -% * f.'Vr'
s» '-' V' i r *v H J| •- , A ^ '.•5 fh <*'- ,- . . , - ' -a, ' *4- ,-V- .'«• ".'viL • V , . * ' • . ,V 1- - %. \ '.V. V '• r' V , _
\ v - i X c K i V r \ & >: ••*• ^ ... ''»• • ,, "«*. • A .f:', , *5.'v •» .t ,
- *, \ . . / > ' . t4 •" '•
A v -' ',. .'V>? .y-Tr. •> - •, •:
:•„% •:•,;••;'>:1v.-t v-,
\ ':'v -A M - rv* * ';r
: J V - ' * j,". (fc*'1 ; \ 'il
. . , "• a i,/ .•• r * "* 'i . <: * v:' % *\ v-/' *' - \ "
frs'-^ " - - ',.-.V'.ri •*'••' ;'.• ..v • ' "' ^ ' >v. 1 ' 4--* "-' ^ • ,•>••>•-.. ., " -r,r •'¥*'• 4v.;r , v//,- ;...- ... , »
Vw?"4"* . - •:• ' •" 'ii.'- x'l ri» -;A •., . fe "'V..,, ^
Fig. 14.—Autoradiogram of Control Skin Tissue— Germinal Epithelial Cell (51'000 x).
the germinal epithelial cells which were close to a blood
vessel. An exact _in vivo location of radiophosphorus could
not be determined. For a more complete discussion of radio-
phosphorus uptake by skin and other tissues, the reader is
referred to the articles listed at the end of this chapter
(1-8). Noteworthy is the close correlation between the ex-
perimental results obtained in this study by autoradiography
and results obtained by others with different methods.
29
- v.C** -vTi: w. • r - ,»/ < ^ * '1 - .«*
^ -< 1 Sf * .•*'* ' " "vs.:;V
l!, /""
4-
v j*\-
,</*>1 * * 'a'* -i *>""
w ^ * *• £ , •' ' I' " /** •" >£"
• \ N ' , ? A
";-- < , - A t .A '•• ft. „<<f . /
V» ,v ,JS».' w
• * f"
- , • * -• ^ * * - , •
1 t
** *y<
"V-"
• » . * \
. # ••'*' 4 £ ^ / - . • * ' ' ! ; - • £ • ' & ? • • ; * . -\ * - .--k, * .'.• p" ?'><T - V" •. >• > X*. 4 J
* . v V /„ 5 f * £ . .
>>'* i
•V
;i,:l1 * ;s! ' *a/ 7Vi#^v^-*\v ;, . *-\ t* >,v^. ^
•<C T'
« *\J, .. * .>'
/ ' '" ;'
***> '«.SS .>•!>
%** * *fv • i" >. - i . ., ya . • '#.. >'i
.*.;/'V
4: •$, ^ -
* . - * f ' > . • ? > . ' '
K ' ;v< •. :r;
v fC' • i p \ ' • • ..•••4'v
$ tr i f , £{ •.
*| % ! i , y r = ' \
H5- s"
V.
• ®i " • *i~. ' jr- ; i' / **it y - " " /, V. - • V.?. * , # "
*''
V v- ' ;;''' , ,, 'i-r
x': •'
?:-v, • .
**•* - f pr" fT '-;'" "v" ' t-i'"'*>'• ' f ,l <"''
Y-; 4' :-"V' "4 - --
Fig. 15-—Autoradiogram of Skin Germinal Epithelium from a Mouse Sacrificed Two Hours after Injection with 3 3P (31,000 x).
CHAPTER BIBLIOGRAPHY
1. Irving7 James T., Dynamics and Function of Phosphorus in Mineral Metabolism, Vol. II, edited by C. L. Comar and Felix Bronner, New York, Academic Press, 1964.
2. Katchman, B. J., "Phosphates in Life Processes," in Vol. II of Phosphorus and Its Compounds, edited by J. R. Van Wazer, New York, Wiley (Interscience), 1961, 1281-1543•
5- Kawin, B. and R. F. Palmer, "Absorption and Distri-bution in Rats of Radioactive Phosphorus Biologically Incorporated in Food," Nature, CLXXXI (May, 1958), 127.
4. Levenson, S. M., M. A. Adams, H. Rosen, and F. H. L. Taylor, "Studies in the Phosphorus Metabolism in Man. III. The Distribution, Exchange and Excretion of Phosphorus in Man Using Radioactive Phosphorus (p32) as a Tracer," Journal of Clinical Investigations, XXXII (June, 1953), 497.
5. McElroy, William D. and Bentley Glass, Phosphorus Metabo-lism, Vol. II, Baltimore, Johns Hopkins Press, 1952-
6. Scholes, V. E., personal communications, Department of Biology, North Texas State University, Denton, Texas, 1969.
7 . Scholes, V. E., A. A. Werder and C. G. Huggins, "Incor-poration of p32 into Phosphatide-Peptide Fraction of Normal and Neoplastic Mouse Epidermis," Proceedings of the Society for Experimental Biology and Medicine, CVII (April, 1961).
8. Schneider, W. C. and H. L. Klug, "Phosphorus Compounds in Animal Tissues. IV. The Distribution of Nucleic Acids and Other Phosphorus-Containing Compounds in Normal and Malignant Tissues," Cancer Research, VI (June, 1946), 691-694.
CHAPTER IV
DISCUSSION
Liquid Scintillation Study
This study points out the movement of radiophosphorus
from the excised tissue to the electron microscope pre-
parative solutions. This movement denotes a large amount
of radiophosphorus diffusion occurring in excised tissue,
making it difficult to locate by autoradiography. This is
not unique to phosphorus, but is true of any small, soluble
?
compound. Many workers have attempted to eliminate diffusion
of small radioisotopes through various methods of freezing
the excised tissue (1, 15/ 17)- These attempts met with
varying degrees of success when locating radioisotopes at
the light-microscope level. Even at this level, morpho-
logical changes, resulting from freezing and thawing, are
frequently severe enough to make location of the tracer•
difficult. At the ultrastructural level, morphological
changes are usually more acute. To date, no one has re-
ported a completely satisfactory solution to the problem
of diffusion of small radioisotopes.
352
In this study,, techniques already employed in electron
microscopy were used to inhibit diffusion of radiophosphate
in excised tissue. A phosphate buffer was of significant
help in inhibiting diffusion of 33P, as indicated in Table
II. This is probably due to the smaller diffusion gradient
between a phosphate solution used as a pH buffer and a non-
phosphate solution used as a pH buffer. Since leaching prob-
ably bears a functional relationship to the amount of diffu-
sion, it can be assumed that considerable diffusion occurs
in excised tissue during its preparation for electron micro-
scopy. The liquid scintillation count study thus gave a
quantative idea of how much radiophosphorus diffused out in
the autoradiographic study.
The liquid scintillation study also provides information
concerning the relationship between the amount of radiophos-
phorus uptake by the different tissues and their rates of
metabolic activity. The results of this study show that the
more metabolically active the tissue is, the greater amount
of radiophosphorus uptake. For example, liver tissue, which
is the most active of those studied, incorporates radiophos-
phorus to a greater extent than skin, which is known to have
a relatively low rate of metabolic activity. The great
amount of incorporation of radiophosphorus into the liver
55 r
cannot be explained on the basis of metabolic activity alone,
however. It is well known that the liver serves as a tem-
porary storage organ for inorganic substances such as phos-
phorus. This accounts for the large amount of radiophosphorus
taken up by the liver. The amounts of radiophosphorus incor-
poration in the skin, muscle, and kidney, as well as in the
liver, reflect the known relative metabolic activity of these
organs. Thus, in this study, a good correlation is shown by
all the tissues between radiophosphorus uptake and rate of
metabolic activity.
The liquid scintillation studies also provide informa-
tion concerning the relative loss of radiophosphorus by tis-
sues during their preparation for the electron microscope.
For example, the two-hour liver samples retain a smaller per-
centage of the original radiophorus than the other two-hour
samples. One possible explanation of this finding can be
given on a purely physical basis. The concentrating factors
present in living cells no longer function during and after
fixation. This being true, the amount of radiophosphorus
lost would depend upon the diffusion pressures found -in the
specific system involved. For example, the large per cent
(92 f o ) of radiophosphorus lost by two-hour liver tissue can be
partially accounted for by the fact that liver absorbs and
3*
temporally retains much greater amounts than any other organ
at that time. Furthermore, the autoradiographic study indi-
cates that this is found in the aqueous fraction of the liver.
During its preparation for the electron microscope, the liver
loses most of its radiophosphorus to the aqueous fixatives.
Thus, a direct relationship is indicated between the concen-
tration of radiophosphorus in liver tissue and the amount
lost during preparation procedures.
Another possible, explanation as to why tissues lose d.if«
ferent amounts of radiophosphorus to the preparatory solutions
can be made on a biological basis. It is believed that those
tissues which retain a greater absolute amount of radiophos-
phorus after preparation do so because a greater proportion
of radiophosphorus has been incorporated into less diffusable
organic compounds. The results of this study indicate that
this is the case. For example, two-hour tissue samples of
liver contain the greatest absolute amount of radiophosphorus
after these procedures. Next are kidney, muscle, and skin in
descending order. Again, a direct relationship is indicated
between the amounts of radiophosphorus incorporated and re-
tained by each tissue and their rate of metabolic activity.
A question may also be raised concerning why some tis-
sues appear to lose a greater percentage of radiophosphorus
55
t© the electron microscope preparatory solutions with increased
incorporation time^ while others lose less. For example, the
Percentage of radiophosphorus loss in skin tissue is less in
twelve-hour tissue samples than in two-hour samples. The in-
creased retention in skin tissue is probably due to a larger
proportion of organic phosphorus compounds in twelve-hour
samples than in two-hour samples. Again,, the larger organic
compounds of phosphorus are probably less diffusible in the
aqueous electron microscope preparatory solutions than their
inorganic predecessors and are more likely to remain jln situ.
h similar situation exists with liver tissue, although the
percentage amounts of radiophosphorus differ since one of the
functions of the liver is to serve as a storage organ. On
the other hand, there is an apparent increase in percentage
loss or radiophosphorus in muscle tissue samples with in-
creased incorporation time, This may be partially accounted
for by the rapid conversion of inorganic phosphorus into cre-
atine phosphate, which is soluble in aqueous solutions. Never-
theless, this does not account for the increased percentage
loss, and a complete explanation of this phenomenon required
further study. Kidney tissue retains approximately the same
percent of radiophosphorus regardless of incorporation time.
This is probably because the kidney is primarily an excretory
56
r
organ. Thus, even though it is a rapidly metabolizing organ,
most of the radiophosphorus it contains at the time of sacri-
fice is apparently diffusible in aqueous solutions. This
could be inorganic rediophosphorus in the process of being
excreted.
Emulsion Sensitization Preliminary Study
The second major problem which exists when radiophos-
phorus is used in autoradiography is, again, not unique to
phosphorus, but is true of any radioisotope having an energy
level higher than tritium, i.e., 34S, 1AC, 32P and 33JP. The
problem is one of finding an emulsion which is sensitive
enough to record the passing of high energy beta particles
while providing useful resolution. Generally, the sensitiv-
ity of the emulsion is directly related to the grain size (j),
i.e., the larger the crystal, the greater the sensitivity.
It is unlikely that radioisotopes with an energy level higher
than tritium (approximately 0-0l8MeV) will expose small sil-
ver halide grains, such as Kodak NTE. The emitted beta par-
ticle from atomic decay is unlikely to lose enough energy in
passing through the crystal to cause exposure (l^). This is
based on evidence that the emitted beta particle loses most
of its energy at the end of its path and relatively little,
close to its source (12). In view of this, very fine-grained
37
emulsions, in very close proximity to the radioactive source
(approximately 50 Angstroms), are difficult to expose- This
difficulty can be overcome by using very low energy isotopes,
high levels of radioactivity, more sensitive emulsions, or
any combination of these.
Even though the above discussion concerning the exposure
of small-grained emulsions is accurate, it does not describe
the total picture. Just as several photons are necessary to
produce a latent image when light is the exposing agent,
several beta particles are necessary when radioisotopes are
the exposing agent (4, p. l8l; 2, 10, 16, 18). The major
difference between these two exposing agents is the time in-(
terval between successive photon bombardments and successive
beta particle bombardments on the silver halide crystal. In
the former, it would be a small fraction of a second; in the
latter, it would typically be minutes or hours. When the
specific activity of the radioactive source is high, there
is a very short time interval between several successive beta
particle passages. As a result, a developable latent image
is formed. When the specific activity of the radioactive
source is low, such as found in tissue sections used for high-
resolution autoradiography, the probability of successive
beta particle passages occurring rapidly enough to cause the
38
formation of a developable latent image is almost zero (l4).
It would appear that more sensitive emulsions should be
developed. Unfortunately, most attempts at making more sen-
sitive emulsions have caused unwanted background density.
For example, gold salts, used to sensitize emulsions, help
maintain the latent image from loss of reciprocity effects
(7. 9 ) . However, they are responsible for causing some .
grains, not exposed by the radioisotope, to develop as well.
In another method for sensitization, the emulsion is exposed
to small quantities of light prior to exposure by the radio-
isotope (8, p. 127)» This is the method employed in this
study. The following brief description of the photographic
process indicates, however, that this method is not true sen-
sitization.
Experimental evidence indicates that two atoms of reduced
silver in the silver bromide crystal constitute the minimum
stable latent image (6, p. 110). Several more reduced silver
atoms must be deposited in a latent image speck of the silver
bromide crystal in order to have a developable latent image
(6, p. 99) • In. small-grained emulsions, the electrons, which
reduce the silver ion initially, normally come from conduction
bands in the silver halide crystal (6, p. 10375). These are
immediately replaced by electrons from sulphur, which is
39
found surrounding the proteinacious gelatin (11, 5)• The
developer is then capable of continuing the reduction of sil-
ver by rapidly depositing electrons at latent image sites.
Thus, silver halide crystals having the minimum amount of
silver deposited in latent image specks are developed. Crys-
tals not having a sufficient amount of reduced silver in
latent image specks will not develop in the time allotted
for normal photographic development (6, p. 88).
In this study, silver halide crystals were used which
had been exposed to enough light to produce a stable latent
image. This image was not developable, as such, but provided
a foundation upon which further development of the latent
image could occur without loss of reciprocity between bombard-
ments of the intermittent beta particles. It was determined
experimentally that short, bright flashes of light work bet-
ter for this end than longer exposures of less intense light,
even though the amount of light is approximately the same
(8, p. 128). Long, low-intensity exposures appear to be ir-
regular in exposing grains, because they produce fewer and
larger developable latent image specks on some crystals, but
not on others (8, p. 127). Short, high-intensity exposure
produces more and smaller subminimal latent image specks on
each crystal. Thus, the subminimal pro-exposure of light
40
causes the formation of several pre-latent image centers in
the-silver halide crystal which are not developable until
they enlarge into latent image centers upon further exposure.
Autoradiographic Study
This study shows the general location of radiophosphorus
incorporated into four different living tissues. There is a
correlation between the photographic density of emulsion ex-
posed by the various tissues and the amount of radiophosphorus
incorporated by each tissue as determined by the liquid scin-
tillation study. As discussed previously/ negative chemo-i
graphic effects must be considered when making this correl-t
ation.
The mathematical calculation of autoradiographic reso-
lution with S3P was not attempted in this study because this
calculation can be more accurately determined through the use
of point sources. Nevertheless/ the examination of several
autoradiograms of tissues in the electron microscope confirm
whether the tracer was more frequently located over the aque-
ous fraction of the cell or the organelles. It was concluded
that at two hours after injection, most of the radiophosphorus
is found in the aqueous fraction of all the tissues studied.
In the liver, there was a progressive increase in the number
41
of grains lying over the mitochondria at four and twelve
hours after injection. At the same time, there was a corres-
ponding decrease in the aqueous fractions of liver tissue.
CHAPTER BIBLIOGRAPHY
1. Appleton, T. C., "A Method of Reducing Diffussion by Freezing," Journal of the Royal Microscopic Society, LXXXIII (March, 1964), 277.
2. Berg, W. F., Photographic Corpusculaire, Paris, Scien-tifique:, 1958.
5. Farnell, G. C. and P. G. Powell, "The Effect of Grain Size on Photographic Sensitivity," Journal of Photo-graphic Science, X (April, 1962), 26>T7~
4. Hamilton, J.- F., "Photographic Effects of Electron Beams, X-rays, and Gamma Rays," The Theory of the Photographic Process, third edition, edited by T. H. James, New York, Macmillan Company, 1967.
5. , F. A. Hamra and L. E. Brady, "Motion of Electrons and Holes in Photographic Emulsion Grains," Journal of Applied Physics, XXVII (December, 1956), 874.
6. , and F. Urbach, "The Mechanism of the Formation of the Latent Image, " The Theory of the Photographic Process, third edition, edited by T. H. James, New York, Macmillan Company, 1967-
7. Hamm, F. A. and J. J. Comer, "The Electron Microscopy of Photographic Grains," Journal of Applied Physics, XXIV (December, 1953), 1^97-
8. Hillson, F. J. and E. A. Sutherns, "Disposition of the Latent Image," The Theory of the Photographic Process, third edition, edited by T. H. James, New York, Mac-millan Company, 1967.
9- James, T. H., "The Site of Reaction in Direct Photographic Development. II. Kinetics of Development Initiated by Gold Nuclei," Journal of Colloid Science, III (October, 1948), 447-455.
J] o
10- Maerker, R. E., "Estimation of the Critical Period in Latent-Image Formation by Intermittent Exposures," Journal of Optical Society of America, XLIV (January, 195*0' 8.
11. Matejet, R., "Zur elektrischen Storleitung in Halogen-sibber-Einkristallen," Naturwissenschaften, XLIII (September, 1956), 533-
12. Oldenberg, Otto and Wendell G. Holladay, Introduction to Atomic and Nuclear Physics, New York, McGraw-Hill Book Company, 1967 -
15. Rogers, Andrew W., Techniques of Autoradiography, New York, Elsevier Publishing Company, 1967.
1^. Stock, Jurgen, "Remarks on the Critical Time Period and the Schwarzschild Exponent in Photographic Processes," Journal of the Optical Society of America, XLVI (August, 1956), 17-21.
15. Stumpf, W. E., "Autoradiography with Cryostat Sections," Stain Technology, XXX (October, 1964), 219-
16. Webb, J. H., "Low Intensity Reciprocity-Law Failure in Photographic Exposure: Number of Quanto Required to Form the Stable Sublatent Image," Journal of the Optical Society of America, XL (January, 1950), 3- 13-
17. Wilske, K. R. and R. Ross, "The Use of Freeze-substitution in Autoradiography," Journal of Histochemistry and Cytochemistry, XIII (May, 1965), 38.
CHAPTER V
SUMMARY
This study has shown 33p to be a useful radioisotope
for high-resolution autoradiographic studies. The effec-
tiveness of this radioisotope, however, hinges on the con-
trol of its high energy level and its diffusion in excised
tissue. This study determined the Intracellular location
of radiophosphorus, autoradiographically, by exposing the
emulsion to small amounts of light prior to radiophosphorus
exposure. These studies indicate that radiophosphorus is
first incorporated into the aqueous fraction of living
tissues and from there, incorporated into other subcellular
fractions, such as the mitochondria. Further studies on
radiophosphorus diffusion are indicated, however, if 3Sp is
to become more useful in high-resolution autoradiography.
BIBLIOGRAPHY
Books
Berg, W. F., Photographic Corpusculaire, Paris, Scientifique, 1958.
Gilbert, Frank A., Mineral Nutrition and the Balance of Life, Norman, Oklahoma, University of Oklahoma Press, 1957-
Irving, James T., Dynamics and Function of Phosphorus in Mineral Metabolism, Vol. II, edited by C. L. Comar and Felix Bronner, New York, Academic Press, 1964.
McElroy, William D. and Bentley Glass, Phosphorus Metabolism, Vol. II, Baltimore, Johns Hopkins Press, 1952.
Oldenberg, Otto and Wendell G» Holladay, Introduction to Atomic and Nuclear Physics, New York, McGraw-Hill Book Company, 1967.
Rogers, Andrew W., Techniques of Autoradiography, New York, Elsevier Publishing Company, 1967.
Westermark, E. G. T., I. G. A. Fogelstrom-Fineman and S. R. Forberg, An Approach to the Production of Phosphorus-32 in Millicurie Quantities in Radioisotopes in Scientific Research, Vol. I, edited by R. C. Extermann, New York, Pergamon Press, 1958.
Articles
Apelgot, Sonia and Raymond Latarjet, "Comparison of 'Suicides' of Bacteria Marked by Radioactive Phosphorus J>2 and 53/" International Journal of Radiation Biology, X (February, 1966), 165-175.
Appleton, T. C., "A Method of Reducing Diffusion by Freezing," Journal of the Royal Microscopic Society, LXXXIII (March, 1964), 277-
4 5
46
Bachmann, L., and M. M. Salpeter, "Autoradiography with Electron Microscope. A Quantitative Evaluation," Laboratory Investigation/ XIV (June, 1965)/ 104l-1053»
Caro, L. G. and M. Schnos, "Tritium and Phosphorus-32 in
High Resolution Autoradiography," Science, CXLIX (July, 1965), 60-62.
, and R. P. van Tubergen, "High-Resolution Auto-radiography. I. Methods," Journal of Cell Biology, XV (March, 1962), 173-188.
Farnell, G. C. and P. G. Powell, "The Effect of Grain Size on Photographic Sensitivity," Journal of Photographic Science, X (April, 1962), 26l.
Hamilton, J. F., "Photographic Effects of Electron Beams, X-rays, and Gamma Rays," The Theory of the Photographic Process, third edition, edited" by T. H. James, New York, Macmillan Company, 1967.
, F. A. Hamm and L. E. Brady, "Motion of Elec-trons and Holes in Photographic Emulsion Grains," Journal of Applied Physics, XXVII (December, 1956), 874.
, and F. Urbach, "The Mechanism of the Form-ation of the Latent Image," The Theory of the Photo-graphic Process, third edition, edited by T. H. James, New York, Macmillan Company, 1967
Hamm, F. A. and J. J. Comer, "The Electron Microscopy of Photographic Grains," Journal of Applied Physics, XXIV (December, 1953)/ 1497 •
Hillson, P. J. and E. A. Sutherns, "Disposition of the Latent Image," The Theory of the Photographic Process, third edition, edited by T. H. James, New York, Macmillan Company, 1967.
Katchman, B. J., "Phosphates in Life Processes," in Vol. II of Phosphorus and Its Compounds, edited by J. R. Van Wazer, New York, Wiley (Interscience), 1961, 1281-13^3-
Kawin, B. and R. F. Palmer, "Absorption and Distribution in Rats of Radioactive Phosphorus Biologically Incorporated in Food," Nature, CLXXXI (May, 1958), 127-
47
James, T. H., "The Site of Reaction in Direct Photographic Development. II.- Kinetics of Development Initiated by Gold Nuclei," Journal of Colloid Science,.Ill (Octo-ber, 1948), 447-455.
Levenson, S. M., M. A. Adams, H. Rosen, and P. H..L. Taylor, "Studies in the Phosphorus Metabolism in Man. III. The Distribution, Exchange and Excretion of Phosphorus in Man Using Radioactive Phosphorus (P32) as a Tracer," Journal of Clinical Investigations, XXXII (June, 1955), 497•
Luft, J. H., "Improvements in Epoxy Resin Embedding Methods," Journal of Biophysical and Biochemical Cytology, IX (October, 19 6l), T409-414.
Maerker, R. E., "Estimation of the Critical Period in Latent-Image Formation by Intermittent Exposures," Journal of Optical Society of America, XLIV (January, 1954), 7-
Matejet, R., "Zur elektrischen Storieitung in Halogensibber-Einkristallen," Naturwissenschaften, XLIII (September, 1956), 555-
Mayr, J., "The Use of Nuclear Emulsions to Determine Small Amounts of P33 Present in Samples of p3s," Experientia, XI (April, 1955), 11-
Millonig, G., "Further Observations on a Phosphate Buffer for Osmium Solutions in Fixation," Fifth International Congress for Electron Microscopy, Vol. II, edited by Sydney S. Briese, New York, Academic Press, 1962.
, "A Modified Procedure for Lead Staining of Thin Sections," Journal of Biophysical and Biochemical Cytology, XI (November, 196l), 736-759-
Salpeter, M. M. and L. Bachmann, "Autoradiography with the Electron Microscope. A Procedure for Improving Reso-lution, Sensitivity and Contrast," Journal of Cell Biology, XXII (July, 1964), 469-477.
Schneider, W. C. and H. L. Klug, "Phosphorus Compounds in Animal Tissues. IV. The Distribution of Nucleic Acids and Other Phosphorus-Containing Compounds in Normal and Malignant Tissues," Cancer Research, VI (June, 1946), 691-694. ~ ~~" ~~ ~
48
Scholes, V. E., A. A. Werder and C. G. Huggins, "Incorpor-ation of P 3 2 into Phosphatido-Peptide Fraction of Normal Neoplastic Mouse Epidermis," Proceedings of the Society for Experimental Biology and Medicine, CVII (April, 1 9 6 l ) ,
Sheline, Raymond K., Richard B* Holtzman, and Chang-Yun Fan, "The Nuclide P 3 3 and the P 3 2 Spectrum," Physical Review, XXCIII (September, 1951), 919-923.
Stock, Jurgen, "Remarks on the Critical Time Period and the Schwarzschild Exponent in Photographic Processes," Journal of the Optical Society of America, XLVI (Auqust, 1956), 17-21. ~
Stumpf, W. E., "Autoradiography with Cryostat Sections," Stain Technology, XXX (October, 1 9 6 4 ) , 2 1 9 .
Webb, J. H., "Low Intensity Reciprocity-Law Failure in Photo-graphic Exposure; Number of Quanto Required to Form the Stable Sublatent Image," Journal of the Optical Society of America, XL (January, 1 9 5 0 ) , 3 - 1 3 .
Wilske, K. R. and R. Ross, "The Use of Freeze-substitution in Autoradiography," Journal of Histochemistry and Cytochemistry, XIII (May, 1965)/ 38.
Unpublished Materials
Mace, Robert C., personal communications, New England Nuclear Corporation, Boston, Massachusetts, 1969.
Scholes, V. E., personal communications, Department of Biology, North Texas State University, Denton, Texas, 1969.