Technical considerations on quantitative microradiography on bone

G. Wallgren and K. Holmstrand

188

developmental changes, at least in part, as reflections of specific metabolic activities of the embryo (cf. [4]). The antigens might thus be considered as yolk proteins which are utilized during differentiation. This concept is supported by cell fractionation experiments on unfertilized eggs. These experiments indicated, that both antigens were localized mainly in the yolk granules of the cell. REFERENCES 1. 2. 3. 4.

5. 6. 7.

8. 9.

10.

BECKER, E. L., Federation Proc. 12, 717 (1953). BECKER, E. L., MUNOZ, J., LAPRESLE, C., and LE BEAU, L. J., J. Immunol. 67, 501 (1951). HARDING, C. V., HARDING, D., and PERLMANN, P., Exptl. Cell Research 6, 202 (1954). KAVANAU, J. L., ibid. 7, 530 (1954). OUDIN, J., Compt. rend. 222, 115 (1946). Methods in Med. Research 5, 335 (1952). PERLMANN, P., Expfl. Cell Research 5, 394 (1953). -ibid. 10, 324 (1956). PERLMANN, P. and GUSTAFSON, T., Experienfia 4, 481 (1948). TELFER, W. H. and WILLIAMS, C. M., J. Gen. Physiof. 36, 389 (1953).

TECHNICAL CONSIDERATIONS QUANTITATIVE MICRORADIOGRAPHY G. WALLGREN Department

of Medical

ON ON BONE

and K. HOLMSTRAND

Physics, Karolinska Received

Institutet,

December

Stockholm, Sweden

6, 1956

DURING

recent years the quantitative distribution of mineral salts in bone has been studied by various authors using the technique of quantitative microradiography introduced by Engstrom [2]. The general procedure has been that used by Amprino and Engstrom [l] and later by Owen [3], all of whom eliminated the necessity for determining the thickness of sections by making absorption measurements on closely adjacent structures only, and assuming a constant thickness. They then referred their readings to the absorption

in In

a reference our

larger

current

system bone

areas required

specimens

prepared

which studies,

an accurate for

usually the

consisted

necessity

of

determination

microradiography

is

of aluminium

foils.

investigating

mineralisation

of section thickness, usually

of

the

order

which of

100

over

in bone microns.

This has been done with the aid of a profile microscope to an accuracy of k 2 microns. The thickness of the specimen was measured by cutting the section with a microdissecting knife, and the thickness of the cut edge was then measured in the profile microscope, either on a single point or on equidistant points along the edge. These points could subsequently be localized fairly accurately in the microradiogram. Experimental

Cell Research 12

Quantitative microradiography

on bone

189

“Monochromatic” radiation was used in making the microradiograms, and the photographic density was kept within those limits in which the density response was linear with respect to incident X-ray energy. Hence the reference system could be omitted. Although the method outlined above does not provide optimum conditions for photometry, we prefer to use it since we have found it difficult to eliminate the unevenness of intensity in a large field. We have also found that, due to “inhomogeneities” in the foils, a reference system made of aluminium lacks the accuracy that is inherent in the method itself. A highly stabilized X-ray unit (Philips PW 1010) was used as radiation source and a copper target tube was operated at 20 kV. The radiation was filtered through an 18 micron thick Ni-foil. X-ray spectroscopic analysis of the transmitted beam showed that more than 80 per cent of the radiation was within the range of 1.5-1.6 A, thus corresponding to the wavelength of a comparatively pure Cu Ka emission line (1.54 A). The straight line relationship between actual mass in the different steps of a reference system and the absorption of X-rays in the same reference system further showed that the radiation was sufficiently monochromatic to obey the laws for absorption of strictly monochromatic X-rays. The distance between the target and the sample-film sandwich was 30 cm and provided a relatively even field of illumination. For the wavelength 1.54 A, the photographic density response of the emulsions used (Eastman Kodak Spectroscopic Plates GH 649 emulsion numbers 499 and 502) was found to be linear in relation to X-ray intensity up to a photometric density of 0.7. Microradiograms were developed for five minutes in undiluted D 19 B at 18°C and further processed in routine fashion. Densitometry was performed using a “Zeiss Schnellphotometer” equipped with optics which minimized the influence of the Schwarzschild-Villiger effect. Photometric readings were made on a scale with 1000 divisions, and scale deflections could be read within half a division. In the absorption formula for monochromatic X-rays, Z = Z,=e@‘t (in which Z = transmitted intensity, I, = original intensity, e = base of natural logarithm, ,U = linear absorption coefficient, t = thickness in cm), we now substitute the values for Z and Z,, by densitometric values, so that Z = k log,, (iO/i,) and I, = k log,,, (&/id), where i,, i,, and id represent, respectively, transmitted light intensity in the nonexposed emulsion area, in the sample area, and in the direct beam area, and k is a constant. It is thus possible to solve the value for the absorption index ,IL.~ and, taking the section thickness into account, to compute the linear absorption coefficient. In an effort to analyze the various errors associated with this method, a “physical” model experiment was designed. As a test object we used a sheet of fused quartz one cm in length and 0.5 cm in breadth. One half of this sheet was 161 microns thick and possessed a plane parallel tolerance of less than half a wavelength of visible light. The other half of the sheet was wedgeshaped with an inclination of 161 to 50 microns in 5.0 mm. The reproducibility of the densitometric method outlined above over an area with constant thickness was computed from the put values from 100 absorption measurements on the plane part of the sheet on one single microradiogram. The reproducibility with slight changes in photographic density was computed by making 10 measureExperimental

Cell Research 12

G. Wallgren and K. Holmstrand

190

ments on each of ten different microradiograms varying in photographic density from 0.4-0.6. The error associated with the combination of densitometry and the determination of thickness on a given point was calculated as the square-root of the sum of the squared standard deviations of the two independent methods, both giving values having a normal distribution. The accuracy of the determination of thickness and subsequent absorption studies on equidistant points was calculated by applying this technique on the wedge-shaped part of the sample with ten observations on ten different plates, the thickness ranging from 150-50 microns. Finally the smallest detectable mass difference was estimated by registering the smallest change in thickness of the quartz wedge which gave a readable change in the value of i, (half a scale unit). The values of the above studies were all shown to have a normal distribution. The densitometer slit was opened up to include an area of 900 microns2 at a magnification of x 33. I

TABLE

Reproducibility Number observations

Densitometry

Linear absorption coefficient mean value

Standard deviation

Standard deviation per cent of mean

on constant

thickness 161 microns Densitometry on constant thickness, varying density Densitometry + local thickness determination, average thickness of 100 microns Densitometry + thickness determination on equidistant points, thickness varying from 50-150 microns

100

80

+0.70

0.90

10 x 10

81

* 1.30

1.42

100

81

21.66

1.66

10 x 10

81

+2.60

3.25

Sensitiuify

Number of observations

50 50 50

Average thickness in microns

Mean value in microns of smallest detectable difference in mass at optimal conditions

150 100 50

1.28 1.06 0.85

Standard deviation

0.45 0.32 0.23

hfean value in per cent of total mass 0.55 1.06 1.70

Transmission

0.30 0.44 0.62

The values recorded are listed in Table I. The systematic errors associated with the microradiographic method due to impure radiation were calculated from the mean value of ten mass determinations made on the plane part of the quartz sheet Experimental

Cell Research 12

Quantitative microradiography

on bone

191

with strictly monochromatic radiation and with the intensities recorded directly in a Geiger Muller tube fed to a rate meter and recorder. It was found that the values obtained by the microradiographic method were somewhat too high, being of the order of + 5 per cent. It is evident from the table that the microradiographic method for mass determination used by the authors is capable of determining differences in mass of k 1.1 per cent at an average thickness of 100 microns. This figure is only slightly higher than the reproducibility of the densitometry procedure which is rt 0.9 per cent. The method of thickness determination used by the authors to get absolute values for mineralization of bone at an average thickness of 100 microns somewhat decreased the accuracy of mass difference determination. Thus depending upon whether the thickness was measured on easily defined structures or along equidistant points, the smallest detectable difference in mass was calculated to be f 1.66 or t 3.25 per cent, respectively. REFERENCES 1. AMPRINO, R. and ENGSTRBM, A., Acta Anat. 15, 1 (1952). 2. ENGSTR~M, A., Acta Radial. Sup@. 63 (1946). 3. OWEN, M., J. Bone Joint Surg. 38 B, 762 (1956).

THE

EFFECT OF THYROXINE ON THE SWELLING MITOCHONDRIA ISOLATED FROM NORMAL AND NEOPLASTIC LIVERS P. EMMELOT

Department

of Biochemistry,

and C. J. BOS

Antoni van Leeuwenhoek-Huis, The Netherlands Amsterdam, The Netherlands Received

OF

December

Cancer Institute,

20, 1956

IN contrast to the mitochondria isolated from rat diaphragm, heart, spleen, brain and testis [7], rat liver mitochondria have been reported to undergo a profound swelling on addition of thyroxine [5]. It has now been found that mitochondria isolated from primary azo dye-induced liver tumors of mice and rats have largely or completely lost the ability to swell in the presence of thyroxine. Female CBA mice were fed o-aminoazotoluene (o-AAT) and female rats of the inbred strain R Amsterdam received p-dimethylaminoazobenzene (p-DAB) together with a diet deficient in proteins and vitamin B. Mitochondria were isolated (10 min. at 5000 g) and washed twice in 0.30 M sucrose containing 0.001 M ethylenediaminotetraacetate (EDTA) of pH 7.4. Suspensions were made in 0.30 M sucrose buffered with 0.02 M Tris (pH 7.4) and incubated at room temperature in the absence and presence of DL-thyroxine (3 x 10-S M). The spontaneous and thyroxine-induced swellings of the mitochondrial samples, including those of normal animals under various experimental conditions, are listed in Tables I and II as the percentage decrease in the optical densities at 520 m/d. In a few experiments the mitochondria were isolated in the absence of EDTA. 12 - 573701

Experimental

Cell Research 12