Photomicrographic Records-— Hsiao, Fujii, and Fine 
327 
transcribed onto the print. In one of our ex- 
periments, for instance, six cultures of eggs, 
each culture having been treated with a differ- 
ent dose of X-irradiation, were photographed 
successively within 3 min. In this way, a series 
of time-lapse photomicrographs, each bearing 
the time of day when it was taken, was made 
for each sample culture from the zygote to a 
desired stage, such as morula, and the resulting 
photographic prints analyzed and the rate of 
cleavage calculated. In Figure 4 selected exam- 
ples of sequential photographs are reproduced 
to show the stability of the relative position of 
the eggs and their structural change during 
cleavage. 
To identify each egg and estimate its rate 
of cleavage, a tracing of the first photograph 
of each sample is made on transparent paper. 
The drawing of each egg to be followed is 
given a number. By superimposing the tracing 
on the second or any subsequent time-lapse 
photograph of a particular sample culture, the 
eggs can be individually identified. The time 
it takes for an egg in a given sample culture 
subjected to a specific treatment to attain a 
certain developmental stage, (for instance, the 
attainment of the four-cell stage by a zygote 
after acute exposure to 20 roentgens), is deter- 
mined by subtracting the time of fertilization 
from the time the picture was first taken which 
shows the desired stage of the egg. Thus, in 
one experiment the eggs were fertilized at 
12:00 noon, the two-cell stage of egg No. 38 
first appeared in the photograph taken at 1:30 
P.M.; the four-cell stage at 2:15 P.M.; and the 
eight-cell stage at 3: 15 P.M. Thus we can deduce 
that it took 90 min for 1st cleavage, 135 min 
for 2nd cleavage, and 195 min for 3rd cleavage. 
DISCUSSION 
1. Blum and Price (1950) described an ap- 
paratus for following photographically the 
cleavages of sea urchin eggs. They pointed out 
two advantages of the photographic method: 
( 1 ) more accurate timing of the cleavages than 
fixing followed by counting; (2) the cleavages 
of each individual egg can be followed and 
recorded. Blum and Price used two microscopes 
in the inverted position, one for U-V-ray irra- 
diated eggs, and the other for controls. The 
present device requires only one microscope. 
2. In our instrument, observation is not lim- 
ited to one treatment, i. e., one sample of treated 
animals compared with its control. In the pres- 
ent device, a large number of samples, each re- 
ceiving a special treatment, can be compared 
with one or more untreated control or controls, 
using only one photomicrographic apparatus. A 
stand 28-inch long as used in our apparatus, 
can accommodate 20 or more samples at one 
time. The only limit to the number of samples is 
the time required for making one run of pho- 
tography through the whole set of samples, 
and that required for making photographic 
print-analyses later. With this apparatus (as 
with Blum and Price’s) the change in cleavage 
rate due to treatment such as X- irradiatic® 'Can 
be recorded. For example an increase or de- 
crease in the rate of cleavage can be observed; 
or initial retardation followed by recovery is 
made apparent. Similarly, the course of abnor- 
mal development can be followed; some of the 
abnormalities may disappear, others may per- 
sist. Fixing of eggs and later examination can 
not provide a sequential series of recorded 
events, especially recoveries. Furthermore, the 
eggs from a single female can be used in all 
of these samples, thus eliminating individual 
variations resulting from the use of batches of 
eggs from different females. 
3. The instrument described here is one ver- 
sion of our device. Other modifications both in 
the material used and in details of construction 
are possible. For instance, we have also applied 
two coiled springs symmetrically to the micro- 
scope-carrying cart in order to pull the entire 
carriage tightly against the aluminum spacers. 
Another modification is the use of a light cool- 
ing and filtering device. A shallow transparent 
tray containing cold water can be placed on 
the plate glass between the culture samples 
and the light source. 
ACKNOWLEDGMENT 
We are indebted to Dr. Walter R. Steiger 
and Mr. George Yokotaki of the University of 
Hawaii physics department for their advice 
and machine-shop help. 
