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 . — ' ** ^

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if

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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 «

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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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6 • f. .«>

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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 -

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>••*>:* 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.