STUDIES IN HISTOCHEMISTRY
LVII. Determination of the Total Dry Mass of
Human Erythroeytes by Interference
Microscopy and X-ray Microradiography
C H A R L E S N. G A M B L E , M.D., a n d D A V I D G L I C K , Ph.D.
From the Histochemistry Laboratory, Department of Physiological Chemistry, The Medical School,
University of Minnesota, Minneapolis
T h e total dry mass of h u m a n erythrocytes was d e t e r m i n e d by b o t h interference microscopy
a n d x-ray microradiography. T h e d e t e r m i n a t i o n of mass per u n i t area, a n d calculation of
total dry mass per cell were simplified by c h a n g i n g the shape of the cells to spheres which
were then flattened to discs of constant thickness w h e n smeared on glass slides for measu r e m e n t of fixed cells by interferometry, a n d to oblate spheroids w h e n smeared on parlodion-coated slides for m e a s u r e m e n t of fixed cells by x-ray absorption. F r o m x-rav measuremerits of 100 smeared a n d alcohol-fixed cells a m e a n dry mass per cell of 33.7 X 10-12g
was obtained. Interference measurements of 100 fresh cells suspended in isotonic saline
gave a m e a n value of 32.4 X 10-12g while interference m e a s u r e m e n t of 100 smeared a n d
alcohol-fixed cells gave a m e a n value of 30.8 X l0 -1~ g. T h e first two values compare well
w i t h a m e a n corpuscular hemoglobin of 31.2 X l0 -12 g, obtained from dcterminations of
erythrocyte c o u n t a n d hemoglobin, since 95 per cent of the dry mass of the cell is hemoglobin. T h e difference in interference values between the fixed and fresh cells is possibly
due to a difference between the specific refractive i n c r e m e n t of alcohol-denatured hemoglobin a n d t h a t of the unmodified substance. T h e value for the latter was used since t h a t
of the former is unknown.
INTRODUCTION
T h e purpose of this study is to c o m p a r e the values
o b t a i n e d for the total d r y mass of individual
h u m a n erythrocytes by interference microscopy
a n d by x-ray microradiography, a n d to c o m p a r e
these values w i t h those for m e a n corpuscular
h e m o g l o b i n o b t a i n e d from d e t e r m i n a t i o n s of
erythrocyte c o u n t a n d hemoglobin. T h e interference m e t h o d permits m e a s u r e m e n t of d r y mass
in fresh (wet) cells, while the x-ray m e t h o d requires t h a t the cells be dried since the presence of
water contributes to the value obtained.
Previous interferometric studies concerned with
the dry mass of red cells have been those of Hale
(1), who measured the optical r e t a r d a t i o n of
h u m a n erythrocytes, Lagerl6f et al. (2) who measured the formation of heme a n d dry mass per
This work was supported by grants H2028 and RG3911 from the National Institutes of Health, United States
Public Health Service.
Received for publication, January 7, I960.
53
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ABSTRACT
chloride (0.01 M phosphate buffer, pH 7.4) and
washed in 5 changes of the buffered saline solution
by centrifugation at 295 G for 10 minutes. The washed
cell suspensions were then allowed to stand for 48
hours at 4°C. Following this, sphering of the cells was
checked by examination in a hanging drop. A portion
of the ceils was diluted 1 : 50 with the buffered saline
solution, a drop of the cell suspension was placed on a
microscope slide and covered with a coverslip which
was sealed with vaseline, and measurements of the
cells were made by interference microscopy. The
remainder of the cells, diluted 1:10, was used to
make smears which were air-dried, fixed in 95
per cent ethanol for 5 minutes at room temperature,
and rinsed in distilled water. Measurements on
these cells were made by both interference microscopy
and x-ray microradiography. The smears for interferometry were made on scrupulously clean
slides. A drop of distilled water was placed over a
part of each smear and a coverslip rimmed with
vaseline was sealed over it. Smears for microradiography were made on slides previously coated with
2 per cent parlodion. A scalpel blade was used to cut
approximately 10-ram. squares of the smeared
parlodion and they were floated free in distilled
water, mounted on doughnut-shaped discs (6 ram.
inner diameter, 15 mm. outer diameter) of aluminum
foil 30 u thick, and air-dried at room temperature.
Values for mean corpuscular hemoglobin were
calculated from quadruplicate determinations of
erythrocyte count and hemoglobin.
Calculation of Cell Mass
1. Fresh Cells--lnterferometry." For the determination of total dry mass of fresh red cells by interferometry, the sphered cells were suspended in
buffered saline, Fig. 1. The spherical shape was
made apparent by gently pressing on the eoverslip
over a preparation and noting the shape of the cells
as they revolved in the suspending solution.
The total dry mass per cell, Mrb0, is Mrbc =
(m/v)V where m/v is mass per unit volume and V
is total volume. This formula can be written as
occurred.
MATERIALS
(1)
AND METHODS
Preparation of Specimens
Venous blood (human) was immediately placed in
vacutubes (Scientific Products, Evanston, Illinois)
containing sodium sequestrene (disodium ethylenediamenetetraacetic acid). 0.15 ml. of the blood was
then diluted to 15 ml. with buffered 0.15 u sodium
54
where m/a is mass per unit area and t is cell thickness.
If measurements of optical retardation in the interference microscope are made at the center of the
spherical cells, then cell thickness at that point
equals cell diameter, d, and the formula becomes
M,bo=
~=
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U
(2)
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unit area during the development of rat erythrocytes, and Mellors (3) who found a great variation in the dry mass of normal h u m a n erythrocytes
from the same individual. After the present investigation was under way, a report by Ponder (4)
a p p e a r e d in which the concentration of a p p a r e n t
protein in h u m a n red cells was measured by interference microscopy.
The red blood cell is especially well suited to
measurement of dry mass by both the x-ray and
interferometric methods, except for one feature,
its shape as a biconcave disc. Since the determination of cellular dry mass by both methods is dep e n d e n t upon precise definition of cell geometry,
variations in thickness in different parts of the
cell result in difficulties in measurement and calculation. Fortunately, the shape of the red cell can
be easily altered so that x-ray and interferometric
measurements and calculation of total dry mass
become relatively simple. When, e.g., red cells
are suspended in isotonic solution and placed between glass slide and coverslip, they undergo an
immediate transformation from biconcave discs to
spheres without change in volume (5, 6). This
disc-sphere transformation has been shown to be
due to an increase in p H of the suspending solution
due to the alkalinity of the glass slide and coverslip (6), in addition to the absorption of crystalb u m i n from the cells by the glass surfaces (7).
Identical shape changes occur during the early
stages of hemolysis of red cells with slow acting
hemolytic agents (5, 8), and Ponder (4) used a
non-hemolyzing lecithin to alter red cell shape to
be able to calculate the concentration of dry mass
in individual red cells. During the present investigation it was observed that the transformation of
red cells from biconcave discs to spheres also
occurs in suspensions of washed cells in isotonic
solutions. Sphering results when diluted suspensions of washed cells are allowed to stand for 48
hours at 4°C. Approximately 90 per cent of the
cells are then spheres and no visible hemolysis has
1
Interference p h o t o m i c r o g r a p h of fresh sphered h u m a n erythrocytes suspended in isotonic saline.
T h e spherical shape of the cells is indicated by the darker central areas where greater retardation
of light t h r o u g h the full d i a m e t e r of the cell occurs. X 550.
2. Fixed Cells--Interferometry." W h e n spherical cells
a r e s m e a r e d on glass slides they become flattened,
p r e s u m a b l y owing to a d h e r e n c e of the undersurface
of the cells to the slide. Values for m e a n diameters
of fixed a n d fresh cells in the preparations for interferometry in T a b l e I illustrate this. T h e d i a m e t e r
of the s m e a r e d cells, 7.6 4- 0.03 #, even after shrinking
d u e to fixation, is still greater t h a n that, 5.8 4- 0.02
~, for the cells in suspension, Fig. 1, a n d 2. T h e
s m e a r e d cells have the s a m e m e a n d i a m e t e r as t h a t of
s m e a r e d a n d fixed u n a l t e r e d red cells w h i c h are
biconcave discs, 7.5 4- 0.03 u, Fig. 3.
T o d e t e r m i n e the shape of the flattened cells, the
cell images in a negative of an interference photom i c r o g r a p h were spot s c a n n e d with the microdensitometer used by O t t o s o n et al. (10), w h i c h is similar
to that of Lessler a n d C h a r i p p e r (9). Fig. 4 represents
a densitometric tracing along the d i a m e t e r of a
single cell. Relative thickness at points along the
cell d i a m e t e r were calculated from density values,
a n d the central vertical cross-section of the cell
was constructed, Fig. 4. T h e flattened cells are discs
with the edges r o u n d e d off. H o w e v e r they differ
little from true discs, (sections of cylinders) a n d there-
TABLE
I
Dry Mass of Human Erythrocytes*
M e t h o d of
measurement
Mean
diameter
Standard
e r r o r of
mean
tt
Alcohol-fixed
cells (x-ray)
F r e s h cells (interferenee)
Alcohol-fixed
cells (interferference)
Uncorrected
(for Av) m e a n
mass/area
Standard
e r r o r of
mean
10-12 g/,u2
Corrected
mean
mass/area
Mean
mass/cell
Standard
e r r o r of
mean
10-12 g/,.2
10-13 g
6 . 7 :~
0.06
1.47
0.02
1.47
33.7
0.4
5.8
0.02
1.80
0.01
1.85
32.4
0.2
7.6
0.03
0.68
0.01
0.68
30.8
0.1
* 100 cells m e a s u r e d by e a c h m e t h o d .
D i a m e t e r b a s e d on m e a s u r e m e n t of 200 cells f r o m m i c r o r a d i o g r a m .
GAMBLE AND GLICK
Studies in Histochemistry. L V I I
55
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FIGURE
Interference p h o t o m i c r o g r a p h of smeared alcohol-fixed sphered h u m a n erythrocytes. T h e even
retardation of light over the entire surface of the cells indicates that they approximate discs of almost constant thickness. Comparison with Fig. 1 shows the increase in cell diameter from flattening.
X 550.
fore it was considered an adequate approximation
to assume a true disc shape for the calculations.
F r o m equation (1)
M~bc =
4
=
4~
(3)
where l/~rrdet is the volume of a cylinder of diameter,
d, and thickness, t. Interferometric measurements
were made at the center of the cells and equation
(3) was used to calculate total dry mass.
3. Fixed Cells--X-Ray : For x-ray measurements the
spherical cells were smeared on glass slides coated
with 2 per cent parlodion. These cells were also
flattened, but less than the smeared cells in the
preparations for interferometry. This is illustrated
in Table I where the mean cell diameter, 6.7 u,
in the x-ray preparations, Fig. 5, can be seen to
be considerably smaller than that, 7.6 ~, for the
fixed cells in the interferometric preparations. This
difference is probably related to the fact that less of
the cell surface adheres to the parlodion film, which
is hydrophobic, so that the cells more closely approximate spheres.
Microdensitometry along the diameter of the
photographic image of a single cell in the primary
microradiogram provided the density tracing shown
in Fig. 6. Construction of the central vertical cross-
56
section of the cell from the density values revealed
it to be an ellipse which, when rotated about its
minor axis, forms an oblate spheroid. If measurements
of density of the images of the cells in the p r i m a r y
microradiogram are taken at the center of the cell
images, then cell thickness, t, at that point is the
minor axis of an oblate spheroid. F r o m equation (1)
ir,,c =
3- \ 2 ]
2 =
V
(4)
where 4/~Tr(d/2)2 t/2 is the volume of an oblate
spheroid with a major axis of d, cell diameter, and a
minor axis of t, cell thickness.
X-Ray Microradiography
The preparation and m o u n t i n g of the reference
system, the microradiography, densitometry, and
calculation of mass per unit area and total dry mass
per cell was that employed previously in this laboratory (10, l l ) , based on the work of Engstr6m
and Lindstr6m (12) and LindstrSm (13). In this
study the x-ray apparatus developed by Engstr6m
and coworkers was used (14). The microradiograms
were made on Kodak spectroscopic film 649 G H by
exposure for 10 minutes at 1.2 kv. and 1.0 ma.
Principal constituents of red blood cells in per
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1960
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FIGURE 2
Interference p h o t o m i c r o g r a p h of s m e a r e d alcohol-fixed u n a l t e r e d h u m a n erythrocytes. T h e biconcave s h a p e of the cells is indicated. C o m p a r i s o n with Fig. 2 shows the s a m e cell diameter. X 550.
cent of total dry weight, were calculated from d a t a
given by W i n t r o b e (15): h e m o g l o b i n a n d m e t h e m o globin, 96.4 per cent, stromal protein, 1.4 per cent,
a n d lipid, 1.36 per cent. Since no values for the mass
absorption coefficients of hemoglobin, m e t h e m o globin, a n d stromal protein are listed in the d a t a of
L i n d s t r 6 m (13), a n d the effect of the slight a m o u n t
of lipid present is negligible, the average mass
absorption coefficient for a n i m a l protein was used.
T h i s appears to be justified by the great similarity in
e l e m e n t a r y composition, on w h i c h t h e mass absorption
coefficient depends, of various types of protein (13).
T h e f o r m u l a for t h e calculation of total dry
mass of the red blood cell by the x-ray m e t h o d
is given by the e q u a t i o n
Mrbc = Ec W 1.32 ~ra~/6
(5)
where Ec is the p h o t o g r a p h i c density at the center
of t h e cells expressed in parlodion equivalents a n d W
is the weight of the parlodion in 10-12 g / ~ . 1.32 is t h e
ratio between the mass absorption coefficients of
parlodion a n d protein at the voltage used. Cell
diameter, d, was obtained by direct m e a s u r e m e n t on
t h e m i c r o r a d i o g r a m at a magnification of 1200
with a m i c r o m e t e r ocular.
Interference Microscopy
A D y s o n interference microscope (Cooke, T r o u g h t o n ,
a n d Simms, Ltd., York, E n g l a n d ) , w h i c h has been
fE
w
hA
O~
~Z
~Q
Z<
WW
a~
1.5
I.I
FIGURE ,~
0.?
>w
~
2
i
.2L9
0
2
4
6
8
Densitometric tracing across t h e diameter of a
cell i m a g e on t h e negative o1 a n interference
p h o t o m i c r o g r a p h of a s m e a r e d alcohol-fixed
sphered h u m a n erythrocyte. T h e figure u n d e r
the curve represents the central vertical crosssection of the cell calculated from the values along
the curve.
GAMBLE AND GLICK
Studies in Histochemistry. L V I I
57
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FIOURE 3
total d r y mass per cell becomes ( 0 . 0 0 1 6 / x ) V or
(0.0016/x) ~'da/6. Therefore,
0.0016 ~.1.~
Mrbc(corr Av) = rnTrd~ + _ _
a6
X
6
= (m/a -I- O.O083d)n'd2/6
I n the case of the fixed cells the m e d i u m was distilled
water, a n d no correction factor was necessary.
RESULTS
AND
DISCUSSION
T h e v a l u e of 32.4 )( l0 -12 g o b t a i n e d for t h e m e a n
total d r y m a s s of fresh e r y t h r o c y t e s b y i n t e r f e r o m e t r y a g r e e s well w i t h a m e a n total d r y m a s s of
33.7 X l0 -12 g o b t a i n e d b y x - r a y a b s o r p t i o n , T a b l e
I. T h i s is in a c c o r d w i t h t h e findings of D a v i e s ,
E n g s t r 6 m , a n d L i n d s t r 6 m (19), w h o o b t a i n e d
close a g r e e m e n t in results f r o m b o t h t h e x - r a y a b s o r p t i o n a n d i n t e r f e r e n c e m e t h o d s for t h e m a s s of a
n u m b e r of biological s a m p l e s . It is also in a c c o r d
w i t h d a t a b y O t t o s o n , K a h n , a n d Glick (10) w h i c h
s h o w essentially t h e s a m e v a l u e s for t h e m a s s o f
r a t m a s t cells as d e t e r m i n e d by b o t h m e t h o d s .
D i f f e r e n c e s in v a l u e s were well w i t h i n t h e error,
-4-5 p e r cent, of b o t h m e t h o d s (13, 16). I n t h c
case of t h e x - r a y d e t e r m i n a t i o n , t h e e r r o r c o n t r i b u t e d b y iron in the r e d cell is negligible since,
FIGURE 5
P h o t o m i c r o g r a p h of a m i c r o r a d i o g r a m of s m e a r e d alcohol-fixed sphered h u m a n erythrocytes. X 690.
58
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THE JOURNAL OF BIOPHYSICAL AND BIOCHE~IICAL C Y T O L O G Y
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VOLUME 8, 1960
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fully described by Davies (16) a n d H a l e (17), was
used. T h e light source was a zircon arc l a m p equipped
with an interference filter transmitting m a x i m a l l y at
5460 A. M e a s u r e m e n t s of optical p a t h difference were
m a d e with a Dyson visual photometer, capable of a
setting reproducibility of 1/~00 of a wave length (16),
with the microscope wedge plates parallel a n d the
interference b a n d s m a x i m a l l y separated to illuminate
the field evenly. Cell diameters were m e a s u r e d with
a micrometer ocular.
T h e basic formula relating optical p a t h difference,
4,, expressed in wave lengths of the green light used,
to cell mass per unit area is rn/a = 4'/x where
x = 100c~, with a representing the specific refractive
i n c r e m e n t of the substance being m e a s u r e d . Since
the dry substance of the red blood cell is almost
entirely hemoglobin, its specific refractive increment,
0.00193, (18) was used.
In the case of fresh spherical red blood cells,
suspended in isotonic saline, the total dry mass per
cell is given by the mass per unit volume, m / a / d ,
times the total volume, V. Because the specific
refractive i n c r e m e n t for h e m o g l o b i n was obtained
with reference to water (18), a correction factor,
Av, for the weight of material in the v o l u m e of
suspending solution displaced by the cell, was
applied to the formula. This correction factor, calculated from the difference in the refractive index
of 0.15 M s o d i u m chloride a n d water at 25°C., is
( 0 . 0 0 1 6 / x ) g m . / m l . (10, 16) a n d the correction in
0.5
ta3
kO
O.5
O (.9
z
o') ra 0.7
Z <
LO LO
123 n." 0.9
n
~IGURE 6
w-c
fYt--
i
0
i
2
L
4
i
6
Densitometric tracing across the diameter of a cell
image in a primary microradiogram of a smeared
alcohol-fixed sphered h u m a n erythrocyte. The figure
under the curve represents the central vertical cross-section of the cell calculated from the values along the curve.
by Ponder (4), a n d support his view t h a t other
protein, in addition to hemoglobin, is present in
the interior of the red cell, possibly in the form of
a n internal framework (4, 5).
T h e m e a n total dry mass per cell of 30.8 X
10-rig for fixed red cells, d e t e r m i n e d by interference microscopy, is lower t h a n t h a t o b t a i n e d
for fresh cells by the same method, or for fixed
cells by x-ray absorption. This difference m a y
result from a difference between the values for the
specific refractive i n c r e m e n t of native a n d alcoholfixed hemoglobin. T h e value of the latter is unk n o w n a n d t h a t of the native substance was used.
F r o m calculations of cz based on experimental
data for other dried protein, the m e a n total dry
mass per fixed cell, calculated with cz for dilute
hemoglobin solutions, would be expected to be
lower t h a n t h a t for the fresh unfixed cell (16).
The authors are indebted to Dr. Leonard J. Greenberg for many helpful discussions and to Dr. Tatiana
Ivanov for valuable technical assistance.
REFERENCES
1. HALE, A. J., Proceedings of the Physiological
Society, J. Physiol., 1954, 125, 50.
2. LARGERLOF, B., THORELL, g., and AKERMAN, L.,
Exp. Cell Research, 1956, 10, 752.
3. MELLORS, R. C., Texas Rep. Biol. and Med.,
1953, 11, 693.
4. PONDER, E., Nature, 1959, 183, 1330.
5. PONDER, E., Haemolysis and Related Phenomenon, New York, Grune and Stratton, Inc.,
1948.
6. FURCHGOTT,R. F., J. Exp. Biol., 1940, 17, 30.
7. FURCHOOTT,R. F., and PONDER,E., J. Exp. Biol.,
1940, 17, 117.
8. GITTER, S., KOCHWA, S., DANON, D., and DEVRIES, A., Arch. int. pharmacod., 1959, 118,
350.
9. LESSLER, M. A., and CHARIPPER, H. A., Science,
1949, 110, 429.
10. OTTOSON, R., KAHN, K., and GLICK, D., Exp.
Cell Research, 1958, 14, 567.
11. OTTOSON, R., and GLICK, D., Exp. Cell Research,
1959, 16, 88.
GAMBLE AND GLICK Studies in Histochemistry. L V l l
59
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by calculation, it c a n a c c o u n t for only 0.6 per
cent of the total energy absorbed. If the hemoglobin is assumed to be fully oxygenated, this oxygen accounts for a n additional 0.5 per cent.
C o m p a r i s o n was m a d e of the total dry mass of
the red cells with m e a n corpuscular hemoglobin,
M C H , calculated from quadruplicate d e t e r m i n a tions of erythrocyte count, 4.81 X 106/mm. s, a n d
h e m o g l o b i n concentration, 15.0 gm. per cent.
Since very close to 95 per cent (15) of the d r y
mass of the h u m a n erythrocyte is hemoglobin,
0.95 Mr~c ~-- M C H . Values estimated for M C H
of 32.0 X 10-12g from x-ray absorption a n d of
30.8 ;< 10-12 g from interferometry agree well with
value of 31.2 )< 10-1~ g for actual M C H .
T h e m e a n difference between m e a n total dry
mass per cell a n d M C H represents the mass of
d r y material other t h a n h e m o g l o b i n in the red
cell. Values of 2.5 X 10-12g a n d 1.2 X 10-12g
for x-ray a n d interferometry, respectively, are in
fair a g r e e m e n t w i t h t h a t of 3.1 )< 10-12g found
12. ENOSTR/SM, A., and LINDSTROM, B., Biochim. et
Biophysica Acta, 1950, 4, 351.
13. LINDSTR/SM, B., Acta Radiol., Suppl. 125, 1955.
14. ENCSTRSM,A., LUNDBERO,B., and BEROENDAHL,
G., J. Ultrastruct. Research, 1957, 1, 147.
15. WINTROBE, M. M., Clinical Hematology, Philadelphia, Lea & Febiger, 4th edition, 1956.
16. DAVIES, H. G., in General Cytochemical Meth-
ods, (J. F. Danielli, editor), New York, Academic Press, Inc., 1958, 57.
17. HALE, A. J., The Interference Microscope in
Biological Research, London, E. & S. Livingstone, Ltd., 1958.
18. ADAIR, G. S., OGSTON, A. L., and JOHnSTOn,
J. P., Biochem. J., 1946, 40, 867.
19. DAVIES, H. G., ENGSTROM, A., and LINDSTROM, B., Nature, 1953, ]72, 1041.
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