LOCAL REDUCTION
OF S P I N D L E
IN LIVING NEPHROTOMA
SPERMATOCYTES
MICROBEAM
ARTHUR
FIBER BIREFRINGENCE
SUTURALIS
INDUCED
(LOEW)
BY U L T R A V I O L E T
IRRADIATION
FORER,
Ph.D.
From the Department of Cytology, Dartmouth Medical School, Hanover, New Hampshire. Dr.
Forer's present address is Biological Institute of the Carlsberg Foundation, Copenhagen, Denmark
ABSTRACT
Irradiation of the mitotic spindle in living Nephrotoma suturalis (Loew) spermatocytes with
an ultraviolet microbeam of controlled dose produced a localized area of reduced birefringence in the spindle fibers. The birefringence was reduced only at the site irradiated, and
only on the spindle fibers irradiated. Areas of reduced birefringence, whether produced
during metaphase or during anaphase, immediately began to move toward the pole in the
direction of the chromosomal fiber, even though the associated chromosomes did not
necessarily move poleward. Both the poleward and the chromosomal sides of the area of
reduced birefringence on each chromosomal fiber moved poleward with about the same,
constant, velocity. O n the average, the areas of reduced birefringence moved poleward with
about the same velocities as did the chromosomes during anaphase. The area of reduced
birefringence was interpreted as a region in which most, though not necessarily all, of the
previously oriented material was disoriented by the irradiation. The poleward movement of
the areas of reduced birefringence indicates that the spindle fibers are not static, nonchangeable structures. The poleward movement possibly represents the manner in which the
birefringent spindle fibers normally become organized. All the experiments reported were on
primary spermatocytes which completed the second meiotic division subsequent to the
experimentation. Since both the irradiated and the control cells completed the two meiotic
divisions, the movement and irradiation effects studied in the first division were nondegenerative.
INTRODUCTION
Though there has been much cytological work
done on spindles (see E. B. Wilson, 1928; Schrader,
1953), the achromatic spindle fiber components
were not conclusively shown to exist in living cells
until 1953 (Inou6, 1953; see Schrader, 1953, and
Mazia, 1961, for reviews). Using a highly sensitive
polarizing microscope, Inou6 showed that the
chromosomal fibers (from the chromosomes to the
poles), continuous fibers (from pole-to-pole), and
asters are present in living cells, the birefringent
fibers corresponding exactly to the appearance of
the fibers seen in the best fixed and stained preparations (Inou6, 1953; Schrader, 1953). Though
spindle fibers do exist in living cells, and though
many theories attribute to these fibers a major
role in the movement of chromosomes during
95
mitosis (Schrader, 1953; Dietz, 1958; C)stergren
et al., 1960; Mazia, 1961; Inou6, 1964; Roth,
1964), there is no direct evidence that the spindle
fibers have such a role, and there is only scant
knowledge of the physical and chemical nature of
the fibers themselves. Most of the evidence for their
role in chromosome movement is circumstantial,
relying on the facts that kinetochores are necessary
for normal anaphase movement to occur (Cornman, 1944; Ris, 1949; Schrader, 1953; Mazia,
1961), and that during prometaphase movement
kinetochores are often stretched in the direction
of the movement (Hughes-Schrader, 1943, 1947;
Cooper, 1951; Dietz, 1956; Bajer and Mol~-Bajer,
1963; Nicklas, 1963); also, various experimental
agents which destroy spindle structure concomitantly stop chromosome movement (Pease, 1946;
Cornman and Cornman, 1951; Inou~, 1964;
Zimmerman and Marsland, 1964).
The best evidence that spindle fibers have an important role in chromosome movement is of the
last kind. Inou~ (1964) showed that the spindle
fiber birefringence disappeared when the temperature was lowered during anaphase, and that
the chromosomes stopped moving when the
birefringence disappeared. Since the chromosomes
did not resume movement until the birefringence
reappeared, he concluded that apparently there is
a direct relationship between birefringence and
movement. Also, Inou~ (1952) showed that the
chromosomes moved toward the periphery of the
cell during colchicine-induced shortening of the
spindle fibers, and that such movement stopped
when the birefringence disappeared, and therefore
that a shortening of the spindle fibers could cause
the chromosomes to move. While these evidences
are suggestive of the interpretations outlined, those
interpretations are not the only ones possible,
because the experimental agents do not affect
the spindle alone. Temperature changes would
affect all the cellular processes. And, since colchicine is applied to the entire cell, colchicine too
could affect components of the cell other than the
birefringent spindle fibers. For example, colchicine
is known to inhibit some dehydrogenases (Gal,
1938), and to alter chromosome structure (Eigsti,
1940), nucleolar structure (Herich, 1963), lysosome and Golgi apparatus structure (Robbins
and Gonatas, 1964), DNA synthesis (LaCour and
Pelc, 1959; Hell and Cox, 1963; Sriramula, 1963),
RNA synthesis (Creasy and Markiw, 1964), cell
nucleotide content (Wang, Greenbaum, and
Harkness, 1963), and muscle excitability (Le-
96
comte, 1949). Thus, while circumstantial evidences do implicate the spindle fibers, the possibility of non-specific action by the experimental
agents prevents unambiguous interpretation of the
data.
The objection of non-specific action can be overcome if one uses an experimental tool which affects
only the spindle, or a part of the spindle, without
affecting other cellular processes. The ultraviolet
microbeam is such a tool (see Zirkle, 1957; Smith,
1964; for reviews). When the ultraviolet microbeam is focused to a small part of the spindle,
ultraviolet light passes through only that part of the
spindle, the cell membrane, and the cytoplasm
above and below the spindle. The irradiation of the
cell membrane and the cytoplasm near the spindle
is unavoidable, but extra-spindle irradiation serves
as a control for effects due to cytoplasmic and
membrane irradiation. Thus, parts of the spindle
can be selectively irradiated, and changes in function specifically due to irradiation of those spindle
parts can be measured. Direct information regarding the role of the spindle fibers in chromosome
movement can be obtained, therefore, by irradiating spindle fibers with a microbeam, while
following the spindle fiber birefringence with a
sensitive polarizing microscope, by following chromosome movement before and after irradiation,
and by comparing chromosome movement in these
irradiated cells with that in non-irradiated cells,
and with that in cells irradiated in extra-spindle
areas. Such experiments have been performed
(Forer, 1964), and this paper is the first of two
papers reporting the results.
This paper describes the behavior of the areas of
reduced birefringence which are produced by
ultraviolet microbeam irradiation; some implications of this behavior to the nature of normal,
non-irradiated spindle fibers are considered in the
discussion. Chromosome movements in these irradiated cells will be described in detail in a subsequent report (Forer, 1964, and manuscript in
preparation).
M A T E R I A L S AND M E T H O D S
Crane flies (Nephrotoma suturalis, Loew)1 of all stages
were maintained in the laboratory using the method
1 I would like to thank Dr. George W. Byers of the
University of Kansas, Lawrence, Kansas, for identifying the species. The stocks derive from a single
female caught by Dr. P. R. Dietz in Durham, North
Carolina in the spring of 1961.
THE JOURNALOF CELL BIOLOGY • VOLUME~5, 1965
Image
i
Analyzer
i
Compensator
Rectified
objective
Strain-free
condenser ~
~
To irradiate •
To focus
k
•
Stage
Reflecting
condenser
)
Cell containing
hexylene glycol
and water
'
iI
Ii
UV mirror
!
i
i
i
!
!
t
!
!
!
I !
t
Polarizer
Filters
I
Lamp housing
<
I
t~___
HBO- 200
light source
of Dietz, (1956 ; and personal communication, 1961 ).2
Last-instar larvae of the proper stage were chosen,
surface-sterilized with 70 per cent ethanol, and then
covered with Kel-F 10 fluorocarbon oil (Minnesota
Mining and Mfg. Co., St. Paul, Minnesota). Each
testis was dissected out under KeI-F 10 and smeared
onto a clean quartz coverslip (A. D. Jones, Cambridge, Massachusetts). 70 # thick Fluorglas (Commercial Plastics, New York) spacers were placed on
the quartz, a clean glass coverslip was placed on top
of that, and the preparation was partially sealed with
dentist's wax (Conger, 1960). Using this method, the
cells completed the 2 meiotic divisions (prophase I
to telophase II) in over /24 of the preparations.
Chromosome behavior in normal cells will be
described elsewhere (Forer, manuscript in preparation); it is similar to that described by Dietz (1956,
1959, 1963) for other species of crane flies. The three
autosomal bivalents (Fig. 2 A1 and Fig. 2 B2) divide
at anaphase, and the daughter dyads move to the poles
(Fig. 13 A1) while the 2 unpaired sex chromosome
univalents (Fig. 13 A2) remain at the equator. The
2 The method for maintaining a laboratory culture of
N. suturalis is described in detail elsewhere (Forer,
1964). Other methods are given in Laughlin (1958)
and Stich (1963). A detailed description of the technique for making living cell preparations is given
elsewhere (Forer, 1964).
FIGURE 1 A schematic diagram of the polarizing microscope-ultraviolet microbeam arrangement. For simplicity, the substage frontsurface mirror between the focusing cell and the
reflecting condenser was omitted from the
diagram.
univalents do not move poleward until the autosomes
have neared the pole. The individual chromosomal
spindle fibers are clearly visible in metaphase and
anaphase with a sensitive polarizing microscope (e.g.,
Fig. 4 A2 and Fig. 13 A 1).
Phase contrast microscope observations were
made with a Carl Zeiss phase contrast microscope
(Model KF124-202), using the 40 X, 0.65 na objective.
Polarizing microscope observations were made with
an American Optical Company (New York) SuperBin Polarizing Microscope with rectified optics
(Inou6 and Hyde, 1957; Shurcliff, 1962, pp. 154155), 3 or with a Model P-42 polarizing microscope4
modified such that a 20 X, 0.5 na straln-free objective (Swann and Mitchison, 1950; Inou6, 1961) was
used as a condenser, a 43 X, 0.66 na rectified objective was used as the objective, a 17 m# retardation
compensator (E. Leitz, New York; see Inou6, 1961)
was inserted between the objective and the analyzer,
and Polaroid sheets (HN-22 from the Polaroid Corporation, Cambridge, Massachusetts) were used as
analyzer and polarizer. An Osram HBO-200 high3 Made available through the courtesy of the Marine
Biological Laboratories, Woods Hole, Massachusetts,
and Dr. Shinya Inou6.
4 Loaned by the American Optical Company to Dr.
Shinya Inou6.
ARTHUR FORER Spindle Fiber Birefringence
97
pressure mercury arc was used as the light source for
observation, and wavelengths other than the mercury
green line (546 m/z) were removed from the b e a m
by a combination of Corning glass filters (No. 4600
and No. 3387, from Corning Glass Works, Corning,
New York), and an interference filter with peak
transmission of 70 per cent at 546 m # (BairdAtomic, Cambridge, Massachusetts).
Photographs were taken with an A O Spencer No.
668 35 m m camera back with compensator lens and
shutter, using KB-17 film ( A D O X Fotowerke,
Frankfurt, Germany). Photographs of living ceils were
taken at various time intervals, distance measurements were made from positive prints at a final
magnification of 1000, and these measurements were
used to make graphs of position versustime. Distances
were measured from one pole chosen as a reference
point, and were measured in the pole-to-pole direction.
The microbeam irradiations were from the condenser side, using one microscope for both observation and irradiation. (A similar system is described
by Inou~, (1964).) For the irradiations the substage
condenser was replaced by an American Optical
50 X, 0.56 na reflecting lens, and a small aluminum
front-surface mirror (2.0 m m X 0.2 ram) was inserted
into the system such that the light from the ultraviolet source was reflected from it and into the reflecting lens (Fig. 1)) Portions of the small mirror were
masked by painting with India ink; in Fig. 4 A3,
for example, the bright area (UV) is visible light reflecting from the unpainted portion of the mirror
and the dark area surrounding it is the painted portion.
The ultraviolet source was a General Electric
5 Such a system has been suggested by Z a m e n h o f
(1943) and used by Inou5 (1964).
Explanation of Figures
B, bivalent
CF, chromosomal fiber
D, dyad
U, univalent
UV, image of the ultraviolet
irradiation source
Figs. ~ A1, ~ C1, l0 B~, and 10 B4 were photographed through a phase contrast microscope. The chromosomes appear dark against the non-granular spindle area (Fig. ~ A1arrows).
The other photographs (Figs. ~, 4, 6, 8, 10, l l , 13, 15) were taken through a polarizing
microscope. The birefringent spindle fibers (e.g., the chromosomal fibers labeled CF in
Fig. 4 A~, and Fig. 13 A1) appear bright or dark against the background, depending on
whether there is additive or subtractive compensation, respectively (Swann and Mitchison, 1950; Inou~ and Dan, 1951; Inou~, 1961). In the polarizing microscope the bivalents
and dyads have low contrast (Figs. ~ Be; 4 A~2; 13 A1; labeled), and they are easiest seen
as the termination of the chromosomal fibers. (The kinetochores are, by definition, the
chromosomal position at which the chromosomal spindle fibers terminate.)
All photographs are printed at X 1000. The scale in the lower right corner is: 9.5~ in
Figs. 13 and 15; 10~ in Figs. ~, 6, 8, 10 and 11; and 10.5~ in Fig. ~.
Figs. 3, 5, 7, 9, 1~, 14, and 16 are graphs of distance from the pole on the irradiated side
(ordinate), versus time (abscissa), the times being plotted with respect to the time at
which tile cell was irradiated (UV). In the schematic diagram at the top of each graph,
the solid black area represents the area of reduced birefringence, a black line represents a
chromosomal fiber, an ellipse represents a bivalent, the univalents are not shown, a halfellipse represents a dyad, the pole on the irradiated side is labeled Pb and the areas of
reduced birefringence, the kinetochores, and the opposite pole are labeled with the geometrical objects by which they are represented in the graphs. The geometrical objects
which represent the kinetochores are closed (circles, and triangles), and those which represent the areas of reduced birefringence and the pole on the non-irradiated side are open
(circles, and squares). In the graphs, pole P1 is a horizontal straight line. The points representing the area of reduced birefringence distances from/)1 are connected by a solid curve,
as are the points representing the distances from P1 of the other pole; the points representing the distances of the kinetochores from P1 are connected by a dashed curve.
The vertical arrow in Figs. 3, 5, 9, 1~, 14, and 16 represents the time of dyad separation.
The position of the focused ultraviolet source measured from a picture taken 1.0 to 0.5
minutes before the irradiation is plotted on the graphs at the time of irradiation (UV).
98
THE JOURNAL OF CELL BIOLOGY • VOLUME ~5, 1965
AH-4 lamp with its glass cover removed. The measured relative line energies were as follows:
254 m/z
265 mg
280 m#
297 m/,t
303 m/z
1.5
2
1
2
3.5
(See also Baum and Dunkelman, 1950.)
The rationale and the method used to focus the
mierobeam are discussed in detail in the Appendix.
Describing the procedure briefly, the ultraviolet
wavelengths were removed with a filter, a hexylene
glyeol~ontaining cell was inserted between the
small mirror and the reflecting lens, the reflected
visible image of the mirror was focused onto the
FIGURE ~, Cell 68j~7,C. Irradiated during metaphase. A1 : each of the three autosomal bivalents is labeled
with an arrow. A~, AS: the position to be irradiated (on the chromosomal fiber of the left bivalent) is
indicated by a bracket. A4: The ultraviolet source is labeled UV. B1, B~, BS, C~: The position of the
area of reduced birefringence (on the chromosomal fiber of the left bivalent) is indicated by a bracket.
There is weak birefringence inside the area of reduced birefringence. B~: the three autosomal bivalents
are labeled with arrows, which correspond to the arrows in A1.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, --11
B1, + ~
C1, +10
A~, --7
B~, +~.5
C~, +14.5
AS, --5
BS, + 6
C8, +18.5
A4, --0.5
B4, + 7
C4, +19.5
The area of reduced birefringence moved to the pole, and did not displaoe the pole when it reached the
pole.
ARTHUR FORER Spindle Fiber Birefrlngence
99
specimen, and the filter and the hexylene glycol were
removed before the irradiation (Fig. 1). The focus
was corrected for 275 m#.
The ultraviolet output from the AH-4 lamp was
monitored with a General Electric PV-10 ultraviolet
sensitive photovoltaic cell (Jagger, 1961) used in
conjunction with filters and a microvoltmeter (Leeds
and Northrup, Philadelphia, Pennsylvania). The
irradiation times were controlled, and were adjusted
(between 14 and 20 seconds) to give a constant irradiation dose (energy/area). (The AH-4 output
did not vary much in the first 100 hours of use.)
The total ultraviolet energy incident upon the cell
was measured by placing the circular photovoltaic
cell on the stage of the microscope and defoeusing
the reflecting lens such that the ultraviolet beam
covered 0.9 of the surface of the photovoltaic cell
(Uretz, 1962). The measurements indicate that the
ultraviolet energies at the focus point in air were of
the order of 10 ergs//~2. This heterochromatic ultraviolet dose was used in all irradiation experiments.
The effective wavelengths for the irradiation effects
described are less than 320 mu. This was determined
by prolonged irradiation through a filter which had
zero transmission for wavelengths less than 320 m#.
RESULTS
Ultraviolet m i c r o b e a m irradiation of spindle fibers
in living Nephrotoma suturalis (Loew) p r i m a r y
spermatocytes produced areas of reduced birefringence. E a c h discrete area of reduced birefringence was a b o u t the same size a n d shape as the
image of the irradiation source aperture (Figs.
4, 6, 8, I0, 13, a n d 15), a n d the chromosomal fiber
birefringence on b o t h the poleward a n d chromosomal sides of the area r e m a i n e d essentially u n changed by the irradiation (Figs. 2, 6, 8, 10, 13,
a n d 15). E a c h area of reduced birefringence rem a i n e d localized on the irradiated fibers; the
affected area did not e x p a n d to include non-irr a d i a t e d regions. I r r a d i a t i o n with the same dose
outside the spindle region h a d no effect on spindle
birefringence. T h e continuous fiber hirefringence is
very weak a n d h a r d to detect at the meiotic stages
w h e n spindle fibers were irradiated (Forer, m a n u script in preparation), so the experiments are
concerned mainly with areas of reduced birefringence on chromosomal fibers; however, w h e n
PI
.............~
~
I
O0 i •
I0o
::L
c:
e-e+e ' e e
. . . . . . . . . . ~..e-e.,e . . . . . . . . . . ~ . . . . . v e 4 . . . . e . . . . , o
20o
.u'+
E5
50D
40
-20
-I0n
i
I
+I0
UV
Time in minutes
+2i 0
+;0
I~GURE 3 The graph is for tile cell photographed in Fig. 2. The distances from pole Px of both sides of
the area of reduced birefringence are plotted. Both sides of the area moved poleward with about the
same, constant, velocity while the associated chromosome remained at the equator. (For simplicity, the
kinetochore positions of only the left bivalent are plotted; the other two bivalents acted in the same
manner.)
100
TIlE JOURNAL OF CELL BIOLOGY •
VOLUME 2 5 ,
1965
FIGURE 4 Cell 63h~ft,1. Irradiated during metaphase. Aft: The position to be irradiated (on the chromosomal fiber labeled CF) is indicated by a bracket. A3: The ultraviolet source is labeled UV. A4, A5, A6:
The position of the area of reduced birefringence (on the middle and left chromosomal fibers) is indicated
by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, --8
A4, "1-~
Aft, --5.5
AS, - 0 . 5
A6, + 1 0
A5, +5.5
The area of reduced birefringence moved to the pole, and did not displace the pole.
II 1
::L
.=_
I0
°w'Q qP v~l q~Oo'°'tlJ~e°
•
FIGVRE 5 The graph is for the cell photographed in Fig. 4. The area of reduced birefringence distances from /)1 which are shown
are of the chromosomal side of the area only.
The area of reduced birefringence moved poleward with a constant velocity, while the associated chromosome remained at the equator.
(The other bivalents acted in the same manner.)
" Q i ...... •
o
E
O
-~
a
20-
30"
-]0
!
t
+1~)
I
UV
Time
+~'0
"1-~0
in minutes
continuous fibers were discernible at the time of
irradiation, they too h a d reduced birefringence in
the area irradiated (Figs. 2, 6, 8, a n d 15). T h e
a m o u n t of birefringence r e m a i n i n g in the irradiated areas was not the same in the different experiments (e.g., Figs. 2, 6, a n d 8), even t h o u g h
the incident irradiation dose (energy/area) was
the same in all experiments. T h e area of reduced
birefringence on a n y given chromosomal fiber was
not necessarily perpendicular to the long axis of
t h a t fiber; its angle (with respect to the fiber axis)
d e p e n d e d on the angle of the focused irradiation
source aperture, which was different in different
experiments (Figs. 2, 6, 10, a n d 13).
T h e area of reduced birefringence could be seen
in the polarizing microscope, b u t could not be
distinguished in the phase contrast microscope as
being either lighter or darker t h a n its surroundings
(Figs. 2 a n d 10).
T h e first postirradiation observations were m a d e
at 20 seconds after the irradiation was finished;
at this time the area of reduced birefringence was
visible (Forer, 1964). Therefore, if there is a time
lag between irradiation a n d formation of the area
of reduced birefringence, this figure (20 seconds)
is the u p p e r limit for such a time lag.
Each area of reduced birefringence moved
toward the pole immediately after it was formed,
ARTHUR FoRErt
Spindle Fiber Birefringence
101
F m u a ~ 6 Cell 68j8,C. Irradiated during metaphase. A1, A~: The position to be irradiated (on
the chromosomal fibers of the two left bivalents) is indicated by a bracket. AS: The ultraviolet source is
labeled UV. A4, B1, B~: The position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, - 5 . 5
B1, +2.5
A~, - 4 . 5
AS, - 0 . 5
A4, +5~
B~2, +8.5
BS, +9.5
B4, +18.5
The area of reduced birefringence moved to the pole, and did not displace the pole or tim adjacent notaffected fibers.
a n d it c o n t i n u e d to move until it reached the pole. 6
This poleward m o v e m e n t occurred without exception in each of the chromosomal fiber areas of reduced birefringence studied, even t h o u g h the
chromosomes associated with the same chromosomal fibers did not necessarily move poleward at
t h a t time (Figs. 2, 4, 6, 8, a n d 10), a n d even though
the cell was in m e t a p h a s e at the time of irradiation
(Figs. 2, 4, 6, a n d 8). T h e relation between movem e n t of the areas of reduced birefringence a n d
m o v e m e n t of the chromosomes will be discussed in
detail in a subsequent paper.
6 " M o v e m e n t " is defined operationally: as time
proceeds, the distance from the area of reduced birefringence to the pole changes. The area "moves"
toward the poles.
102
Both sides of the area of reduced birefringence
on each chromosomal spindle fiber moved poleward in the direction of the c h r o m o s o m a l fiber,
a n d b o t h sides of the area m o v e d toward the pole
with a b o u t the same velocity (Figs. 2, 8, 13, a n d
15). I n some cells the reduced birefringence on
different chromosomal fibers moved poleward with
different velocities, a n d w h e n this h a p p e n e d , the
area of reduced birefringence c h a n g e d in shape or
in angle relative to the spindle axis as the area
moved toward the pole (Figs. 10, 11, a n d 15).
T h e areas did not deform the a d j a c e n t unaffected fibers as they moved toward the pole (Figs.
2, 6, 8, 13, a n d 15), nor did they push away the
asters once they reached the pole (Figs. 2, 6, 8, 11,
13, a n d 15). Rather, the size of each area gradually
TIlE JOURNAL OF CELL BIOLOGY • VOLT3ME 25, 1965
•
~,~0
P1
• ,.,-.,-.-o~,~
n
P1
I0
~
44
~
..... at..... om--'*°
ao~.O"" llo"'''°e..... * .....
......
C
q~
~ ........
- -
"o-,
""0
FIGURE 7 The graph is for the cell photographed in Fig. 6. The area of reduced birefringence distances from Px which are shown are
of the chromosomal side of the area associated
with the left bivalent. The area of reduced blrefringence moved poleward with a constant
velocity, while the associated chromosome remained at the equator. (The other chromosomes,
and area of reduced birefringence, acted in the
same manner.)
2050
40
-I0
I
UV
l
+I0
I
,u02
+50
Time in minutes
FIGURE 8 Cell 68h13,B. Irradiated during metaphase. A1, A~: The position to be irradiated (on tile
chromosomal fibers of the two left bivalents) is indicated by a bracket. A3: The ultraviolet source is
labeled UV. A4, B1, B~, B3, B4 : The position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the cell
was irradiated:
A1, --6.5
AS, --5
AS, - 0 . 5
A4, + 2
A5, + 3
B1, +4.5
B~, + 6
BS, +11
B4, +16
B5, +17
The area of reduced birefringence moved to the pole, and did not displace the pole.
decreased after the area reached the pole until
such an area was no longer distinguishable.
The primary interest was in effects on the chromosomal fibers of the autosomes. Areas of reduced
birefringence on 99 such chromosomal fibers were
studied; 39 were in cells in metaphase at the time
of irradiation, and 60 were in cells in anaphase at
the time of irradiation. (Figs. 2, 4, 6, and 8 show
ARTrma FOREa Spindle Fiber Birefrlngenee
103
PI
I0
::L
FIGURE 9 The graph is for the cell
photographed in Fig. 8. The area of reduced birefringence distances from P1
which are plotted are of the chromosomal side of the area on the left bivalent's chromosomal fiber. The area of
reduced birefringence moved to the pole
with a constant velocity while the associated chromosome remained at the
equator. (The other chromosomes, and
area of reduced birefringence, acted in the
o--QO . . . . . . . "
c
. . . o . . . o . . m . o . . o . . . u , . o . , ~ - .....
c
o
,m,o...~ii.
20-
~5
50-
40- 20
I
- io
l
i
I
+1o
I
I
+zo
same manner.)
+30
UV
Time
in m i n u t e s
areas of reduced birefringence in m e t a p h a s e cells,
a n d Figs. 10, 11, 13, a n d 15 show areas of reduced
birefringence in a n a p h a s e cells.) Areas of reduced
birefringence on chromosomal fibers of univalents
also moved toward the pole i m m e d i a t e l y after
being formed.
T h e r e was a c h a n g e in the birefringence of b o t h
the areas of reduced birefringence, a n d the chromosomal fibers immediately adjacent to the areas
of reduced birefringence, as the areas m o v e d tow a r d the pole. T h e birefringence inside each area
of reduced birefringence often increased in magnitude as the area moved to the poles (Figs. 2, 4,
a n d 13). T h e birefringence of the chromosomal
fiber immediately adjacent to each area decreased
in magnitude as the area moved to the pole. ( T h a t
the last statement must be true can be seen from
the following considerations. Since a n area of reduced birefringence moves toward the pole, with
time, the position of the chromosomal fiber adj a c e n t to the area moves toward the pole, with
time. After the area of reduced birefringence has
reached the pole, the consecutive positions along
the fiber, from the initial position of the area of
reduced birefringence to the pole, represent the
time course of the position of the fiber adjacent
to the area of reduced birefringence. Since in these
104
cells the spindle fiber birefringence is strong near
the kinetochores and weaker toward the poles
(Figs. 2, 6, a n d 13), and since after the area
reached the pole the irradiated chromosomal fiber
was indistinguishable from n o n - i r r a d i a t e d fibers,
the birefringence of the chromosomal fiber adjacent to the area of reduced birefringence decreased
in m a g n i t u d e as the area moved to the pole.)
T h e distance between the area of reduced birefringence a n d the spindle pole on the irradiated
side was measured on each p h o t o g r a p h in the series
of photographs of each cell. I n favorable cases the
distances from the pole of b o t h sides of the area
(of reduced birefringence) were measured (Figs. 3
a n d 16), b u t in general the distances from the pole
of only the side of the area closest to the chromosomes were measured. T h e chromosomal side of a n
area could be followed until it was 3 to 4 /~ from
the pole, b u t because of the increasing birefringence inside the area, because t h e c h r o m o s o m a l
fibers have less birefringence near the poles, a n d
because the chromosomal fibers converge toward
the poles, it was difficult to distinguish the area
after this point.
T h e poleward velocity of the area of reduced
birefringence on each chromosomal fiber was calculated from t h e distance measurements. T h e dis-
T H E JOURNAL OF CELL BIOLOGY ' VOLUME ~5, 1965
tance of each area from the pole was plotted with
respect to the time elapsed since the fiber was
irradiated, and the slope of this curve is the poleward velocity.
Each area of reduced birefringence moved poleward with a constant velocity (Figs. 3, 5, 7, 9, 12,
14, and 16), but different areas did not necessarily
move with the same velocity, even in the same cell
(Figs. 10, 11, and 15). The distribution of the poleward velocities of the areas (of reduced birefringence) is shown in Fig. 17 A, shaded, for cells irradiated before anaphase, and in Fig. 17 B, shaded,
for cells irradiated during anaphase. The anaphase
poleward velocities of chromosomes in non-irradiated (control) cells adjacent to the irradiated
cells is the non-shaded area of the same figures.
(The chromosome velocities were measured in the
same way as were the area of reduced birefringence
velocities.) These comparisons show that after
irradiation either in metaphase or in anaphase, the
poleward velocities of the areas of reduced birefringence are, on the average, about the same
as the anaphase poleward velocities of chromosomes in non-irradiated control cells. Before making statements regarding the equivalence of area
of reduced birefringence velocities and chromosome velocities, the velocities of individual areas of
reduced birefringence should be compared directly
with the velocities of the chromosomes associated
with the same fiber, or with the velocities of individual control chromosomes. When this is done
(Forer, manuscript in preparation), it is seen that
the velocities of the areas are often different from
FIGURE 10 Cell 63L7,4. Irradiated during anaphase. A1, A~: The position to be irradiated (across the
entire half-spindle) is indicated by a bracket. A3: The ultraviolet source is labeled UV. A4, BI: The
position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, - 9
A~, --7
A3, --0.5
A4, +1.5
B1, +3.5
B~, +4.5
B3, +16.5
B4, +19
The area of reduced birefringence moved to the pole and did not displace the pole. In A4 the chromosomal side of the area of reduced birefringence makes a straight line; in B1 the chromosomal side of the
area of reduced birefringence does not make a straight line, because the areas on different fibers moved
poleward at different rates.
ARTHUR FORER Spindle Fiber Birefringenee
105
FIGURE 11 Cell 63hS,A. Irradiated during anaphase. A1, A~: The position to be irradiated (across the
entire half-spindle) is indicated by a bracket. A4: The ultraviolet source is labeled UV. B1, B% B4: The
position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, --8
B1, +4.5
A~, --5.5
B~, +5.5
.4.8, --4.5
B8, + 1 3
A4, - 0 . 5
B4, +16.5
AS, + 4
B5, +~1
The area of reduced birefringencc moved to the pole and did not displace the pole. In A5 and B1, the
chromosomal side of the area of reduced birefringence makes a straight line; in B3, and B4 the area of
reduced birefringencc does not make a straight line because the areas on different fibers moved poleward
at different rates.
the velocities of the chromosomes, by as m u c h as a
factor of 3. These results will be given in detail in a
subsequent report.
I n the experiments reported here, both the irradiated cells a n d the non-irradiated control cells
completed the two meiotic divisions. (In five cases
the d a t a include irradiated cells which did not
complete division II. In these cases there was
indication of p r e p a r a t i o n for division II such as
aster formation, or spindle formation, a n d the
data were not in variance with other cases where
the irradiated cells completed the second division.)
It is necessary to follow the cells t h r o u g h the completed second division to ensure that the movements observed in n o n - i r r a d i a t e d cells were n o r m a l
a n d not degenerative, a n d to ensure t h a t the differences between the irradiated and the control cells
106
were not due to lethal or semilethal effects of the
irradiation (e.g., U r e t z a n d Zirkle, 1955; Izutsu,
1961; Bajer a n d Mol~-Bajer, 1961). Since the ceils
completed a second division, the movements and
irradiation effects studied in the first division were
non-degenerative.
DISCUSSION
T h e birefringence of the irradiated p a r t of a spindle
can be greatly reduced without c h a n g i n g the birefringence of other parts of the spindle. T h o u g h this
is not the first time such results have been reported
(see Inou~, 1964), most ultraviolet m i c r o b e a m
workers report t h a t the entire spindle structure
disappears after irradiation, even after irradiation
of the cytoplasm (Zirkle, 1957; Smith, 1964). T h e
difference between the findings of those workers
T H E JOUnNAL OF CELL BIOLOGY • VOLUME ~5, 1965
and the localized disappearance reported here is
most likely due to a difference in dose rather than
a peculiarity of crane fly spermatocytes, for the
following reasons: (a) While cytoplasmic irradiation of Tradescantia stamen hair cells could cause
the entire spindle structure to disappear, Otroshchenko and Sakharov (1964) showed that this
did not happen at lower doses, and that depending
on the dose used the spindle might or might not
disappear. (b) Inou~'s results on Haemanthus endosperm (Inou6, 1964) are similar to those on Nephrotoma, yet it had been reported by other workers
that cytoplasmic irradiation destroyed the entire
Haemanthus spindle (Zirkle, Uretz, and Haynes,
1960). (c) The entire Nephrotoma spindle birefringence will disappear if the dose (energy/area) used
for irradiation of spindle fibers is three times that
used here (Forer, 1964).
Since the birefringence was changed only at the
site of the microbeam irradiation, experiments
were performed to test the role of the birefringent
spindle fibers in chromosome movement. These
results will be given in detail elsewhere. In this
paper we will consider only the movement be-
havior of the areas of reduced birefringence, and
the implications of this behavior toward the nature
of normal (non-irradiated) birefringent spindle
fibers. Since such inferences depend greatly on the
exact nature of the area of reduced birefringence,
I will first discuss the nature of the areas of reduced
birefringence, and then discuss some implications
of the behavior of these areas to the properties of
non-irradiated spindle fibers.
Nature
of the
Areas
of Reduced
Birefringence
Material is birefringent when the refractive
index for light whose electric vector is polarized in
a certain direction is different from the refractive
index for light polarized in a perpendicular direction. In an area of reduced birefringence the
refractive indices are roughly the same for all orientations of the electric vector. The ultraviolet microbeam could cause the birefringence to be reduced (a) by causing the molecules which were
previously oriented to become disoriented, or (b)
by changing the intrinsic birefringence of the
oriented molecules without greatly disorienting
them, either through cross-linking or denaturation,
[]
P1
I
....*"i~*.........
6""
IO---*---*-----~--°"°- ~--Q • . . % ° • " * ~ ~ ' o " ~ " ' •
c
.......
2o• 0
"'"-..
•
.
50-
-10
-20
+I0
+ 0
+50
+40
UV
Time in minutes
FIGURE le The graph is for the cell photographed in Fig. ll. The area of reduced birefringence distances
from /)1 which are plotted are of the chromosomal side of the area on the right dyad's chromosomal
fiber. The area of reduced birefringence moved to the pole with a constant velocity even though the two
associated dyads temporarily stopped moving poleward.
ARTHUR FORER Spindle Fiber Birefringence
107
FmURE 13 Cell 63j10,~. Irradiated during anaphase. A~: The position to be irradiated (on the chromosomal fibers of the two right dyads and the univalents, U) is indicated by a bracket. A3: The ultraviolet
source is labeled UV. A4, B3: The position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to tile time at wtfich the
celt was irradiated:
A1, --6
B1, -4.4.5.5
A~, --5
B~, -4.8
A3, --0.5
B3, ,4.10.5
A4, "4"~
B4, +1~.5
AS, -4-3
B5, .4.15.5
Tile area of reduced bircfringence moved to the pole, and did not displace the pole.
or (c) by both denaturation and disorientation,
or (d) if the spindle birefringence is mostly formbirefringence, by changing the relative volumes
of oriented versus non-oriented material. Information to distinguish between these possibilities might
be obtained by light microscope and electron
microscope studies of fixed and stained areas of
reduced birefringence, by micromanipulator studies of such areas compared with non-irradiated
spindle fibers, by radioactive labeling of spindles
and determining if areas of reduced birefringence
lose label, or by following the movement of granules found in such areas; but such information is
not available at the moment.
However, since the amount of birefringence inside the area increased as the area moved toward
the pole, and since the area deformed neither the
adjacent non-irradiated fibers nor the pole, the
area of reduced birefringence is more likely material which is disoriented rather than material
cross-linked or denatured, for I would not expect
cross-linked or denatured material to gain birefringence as it moved toward the pole, and I would
expect cross-linked or denatured material to push
108
the pole away rather than be "disassembled" when
it reached the pole.
Such an interpretation does not imply that all
the structure or orientation is absent from an area
of reduced birefringence, (a) because weak birefringence is often present inside the area after irradiation (Figs. 2 and 13), implying some remaining oriented material, and (b) because there could
be oriented material in such an area which would
not be detected in the polarizing microscope. This
interpretation does imply that much or most of the
oriented structure was destroyed by the irradiation. Thus, I interpret the area of reduced birefringence to be an area in which most, though not
necessarily all, of the previously oriented material
has been disoriented by the irradiation.
Interpretations of the Behavior of the Areas of
Reduced Birefringenee
In Nephrotoma spermatocytes the birefringence
poleward from the area of reduced birefringence
was unaffected by the irradiation which produced
the area of reduced birefringence (this report).
Inou~ (1964) reported that the birefringence pole-
THE JOURNAL OF CELL BIOLOGY • VOLUME~5, 1965
ward (distal) from the irradiation site disappeared
after ultraviolet microbeam irradiation of chromosomal spindle fibers in Haemanthus endosperm. He
attributed the poleward disappearance of birefringence to the absence of an "organizing center"
poleward from the irradiated site. If the poleward
birefringence is unaffected in Nephrotoma because
of the influence of the centriole acting as an "orienting center," the poleward side of the area of
reduced birefringence must be given "disorienting" attributes as well, for the fiber between the
area of reduced birefringence and the pole decreases in length as the area moves toward the
pole. It is surprising that the centriole would have
an influence in maintaining the birefringence, for
both chromosome movement and spindle fiber
birefringence are normal even when there is no
centriole at the pole of Nephrotoma suturalis spermatocytes (Forer, 1964; this was originally demon-
:.-:-: . . . . . . . . . . . . . .
I0
-
::k
,c
c
o
::iL~::::::::--tw-.m . - L * - ~ -_.,
20-
C3
leA..
-,,[:~j
-
50-
4.0
- I0
I
|
t
+10
I
-I-20
UV
Time
in minutes
FmuRB 14 Tile graph is for tile cell photographed in
Fig. 13. The area of reduced birefringence distances
from P1 which are plotted are of the chromosomal side
of the area on the middle dyad's chromosomal fiber.
The kinetochore positions of the members of both the
middle dyad pair and the left (not-irradiated) dyad
pair are plotted. The area of reduced birefringence
moved to the pole with constant velocity even though
the poleward movement of the two associated dyads
temporarily stopped. Both dyads in the left (not-irradiated) dyad pair moved normally.
strated by Dietz, 1959, 1963 for spermatocytes of
the crane fly, Pales crocata). The role of the centriole
in maintaining the birefringence poleward from
the irradiated site could be directly tested by
microbeam irradiation of chromosomal fibers in
spermatocytes in which the centriole has been experimentally dissociated from the pole.
The poleward movement of the area of reduced
birefringence is defined operationally: as time
proceeds, the distance from the area to the pole
becomes smaller. Such behavior might mean that
there is actual movement of the fiber material to
the pole; i.e. that the birefringent material, and
the non-birefringent material in the area of reduced birefringence, both move to the pole, are
broken down at the pole (i.e., are disoriented and
made non-birefringent), and are then re-cycled,
and that the area of reduced birefringence is just
a marker in this continuously moving system. This
is not the only possibility, however. For example,
it is possible that without some accessory component the oriented spindle fiber material will revert to the disoriented state, and that it is this
component and not the birefringent fiber material
which moves to the pole; with this interpretation,
the ultraviolet inactivates this accessory component, the ultraviolet inactivated component is unable to cause orientation, disorientation ensues,
and an area of reduced birefringence is formed.
The area of reduced birefringence moves not when
non-birefringent fiber material moves poleward,
as in the previous interpretation, but rather when
the non-functional accessory component moves
poleward and displaces the functional component.
As another alternative, it is also possible that no
material moves poleward at all, but rather the area
of reduced birefringence movement indicates a
wave of organization which moves to the pole.
Pease (1946) found that spindle fibers grew
from the chromosomes after removal from the high
pressure which had caused spindle breakdown.
While this might suggest that the area of reduced
birefringence movement is an actual movement of
fiber material, the data presented in this paper do
not rule out the other possibilities. However, if
the poleward movement of the area of reduced
birefringence does indicate movement of the fiber
material, this fiber material must change state, or
orientation, as it moves, for as described in the
Results section, there are changes in the birefringence of both the area of reduced birefringence,
and the chromosomal fiber adjacent to the area,
as the area moves to the pole.
AnTlt~J~, FORER Spindle Fiber Birefringence
109
FIGURE 15 Cell 63h~,1. Irradiated during anaphase. Ae: The position to be irradiated (on the chromosomal fibers of the two left dyads) is indicated by a bracket. A3: The ultraviolet source is labeled UV.
A4, B1, B3: The position of the area of reduced birefringence is indicated by a bracket.
The times of the photographs follow; they are given in minutes with respect to the time at which the
cell was irradiated:
A1, --8
B1, +3
A~, --5.5
A3, --0.5
A4, -{-~
B~, +5
B3, -{-6
B4, -t-16
The area of reduced birefringence moved to the pole and did not displace the pole. The chromosomal
side of the area of reduced birefringence is a straight line in A4, but is not straight in B1, B~, and B3
because the areas of reduced birefringence on the different chromosomal fibers moved poleward at different
rates.
T h e poleward m o v e m e n t of the areas of reduced
birefringence, then, represents a poleward movem e n t of some material, or a wave of organization.
Regardless of which of the interpretations is true,
this indicates t h a t even w h e n the chromosomes
do not move, the spindle fibers are not static, nonc h a n g e a b l e structures, for they can drastically
change their organization (see Inoufi, 1959, 1964,
for reviews). O n e question which does arise, however, is w h e t h e r this poleward movement, or wave,
indicates a d y n a m i c organization which was present before the irradiation, or w h e t h e r the fibers
are really basically static, a n d t h a t in c h a n g i n g the
birefringence the irradiation set in motion a process whereby the changed region is propagated to
the pole. At present there are no d a t a to distinguish
between these two possibilities. It is clear t h a t in
Nephrotoma the spindle fiber organization changes
even without irradiation, for the chromosomal
ll0
fibers gradually increase in width a n d birefringence between nuclear m e m b r a n e b r e a k d o w n a n d
a n a p h a s e (Forer, 1964, and manuscript in preparation; also Dietz, 1963; and Inoufi's evidence for
d y n a m i c equilibrium, 1964), a n d thus t h a t even
before anaphase the organization of the spindle
fibers is not static. But it is not k n o w n w h e t h e r
the normal increase in width a n d birefringence is
at all related to the m o v e m e n t of the area of reduced birefringence, n o r if the area m o v e m e n t is
indeed induced by the irradiation. T h e existence
of m o v e m e n t might be ascertained from the following experiment: Ostergren, Mol~-Bajer, a n d
Bajer (1960) hypothesized t h a t the poleward
m o v e m e n t of spindle inclusions (such as nucleoli,
acentric chromosomes, g r a n u l e s , . . . ) was due to
extra-spindle-fiber " p u m p s , " which acted on
spindle fibers in the same way as on the inclusions.
According to the hypothesis, the poleward move-
THE JOURNAL OF CELL BIOLOGY • VOLUME ~5, 1965
°.~-~----®
PI
iO::t..
ar
-0
-0
,4PO
•
t
P" I w
09"~ i l t ~
O.
~
•
"0
• ~$'Q"
-0
~
•
•
c:
2oo
¢o
O
50-
40
~0
-
a
-I0
[
UV
i
÷ I0
i
i
+20
+50
Time in minutes
FmURE 16 The graph is for the cell photographed in Fig. 15. Tim distances from pole P1 of both sides
of the area of reduced birefringence are plotted for the area on the middle dyad's chromosomal fiber.
Both sides of the area of reduced birefringence moved poleward with about the same, constant velocity,
even though the two associated dyads temporarily stopped moving.
m e n t of the area of reduced birefringence would be
a n actual m o v e m e n t of material, a n d would be due
to the same force which causes the spindle inclusions to move poleward. This can be directly
tested by comparing the m o v e m e n t of a n area of
reduced birefringence with m o v e m e n t of spindle
inclusions in the same cell. If the two always
moved with the same, or a related velocity, this
would imply t h a t the poleward m o v e m e n t of the
area of reduced birefringence was due to the same
m e c h a n i s m as the poleward m o v e m e n t of spindle
inclusions. Since the spindle inclusions move poleward in all stages, such a result would lend credence to the idea t h a t the m o v e m e n t of the area
indicates a p h e n o m e n o n which occurs in all stages,
a n d not one which is induced by the irradiation.
(If the two did not agree, however, it would m e a n
only t h a t the reduced birefringence m o v e m e n t a n d
the inclusion m o v e m e n t did not occur by the same
mechanism, and with this result the experiment
would not give information w h e t h e r or not a flow
or wave in the spindle fibers always occurred.)
I interpret the poleward m o v e m e n t of the area
of reduced birefringence to indicate a process
which occurs even without induction by the irradiation, a n d which represents the way in which the
birefringent spindle fibers are organized from
disoriented material, in conjunction with a n as yet
morphologically unidentified traction element
(Forer, 1964; and m a n u s c r i p t in preparation).
F u r t h e r evidence in support of this hypothesis will
be considered in a subsequent publication.
I n conclusion, the results reported here confirm
the findings of Inou~ (1964) that the birefringence
of local parts of a spindle can be greatly reduced
by ultraviolet m i c r o b e a m irradiation w i t h o u t
affecting the birefringence of n o n - i r r a d i a t e d parts.
T h e area of reduced birefringence is i n t e r p r e t e d
as a n area in which most, t h o u g h not necessarily
all, of the previously oriented material has b e e n
disoriented by the irradiation. O t h e r alternatives
are not ruled out by the data, however.
T h e poleward m o v e m e n t of the areas of reduced
birefringence which occurs in b o t h metaphase a n d
a n a p h a s e p r o b a b l y represents a m o v e m e n t of
material, either of oriented molecules themselves,
or of molecules necessary for orientation. It is also
possible t h a t no such molecules move, b u t t h a t a
AI~THUR FoaELt Spindle Fiber Birefringence
lll
70
A
60,
50
4O
FIGURE 17 The shaded areas are the number of autosomal chromosomal fibers on
which an area of reduced birefringence was
formed (ordinate), versus the poleward velocity
of each such area (abscissa). Fig. 17 A, shaded,
is for areas of reduced birefringence in cells irradiated before dyad separation (during late
prometaphase and early metaphase), and Fig.
17 B, shaded, is for areas of reduced birefringence in cells irradiated after dyad separation
(during anaphase). The non-shaded areas are
the number of dyads (ordinate) versus the
poleward velocity of each such dyad (abscissa)
for dyads in the ~lls used as controls for the
Irradiation ,expernnents.
E
z
30
20
I0
O
i
0.0
0.5
H
|
1.0
o.0
0.5
1.0
V e l o c i t y /.dmin.
wave of organization is propagated along the
fiber. W i t h either alternative the results indicate
that the chromosomal fibers are not static, nonchangeable structures.
T h e poleward m o v e m e n t of the areas of reduced
birefringence is interpreted to indicate a m o v e m e n t
which occurs all the time, and which indicates the
m a n n e r in which the birefringent spindle fibers are
organized. An alternative interpretation is possible,
in which such m o v e m e n t or propagation is induced by the irradiation.
I acknowledge with gratitude the counsel of Dr.
Shinya Inour, under whose sponsorship this work
was performed, and I wish to thank him for his
generous loans of equipment, and his critical comments on this manuscript in various stages of its
preparation. I would also like to thank Dr. Roland
Dietz for instruction on the laboratory care of crane
flies, and for the gift of some Nephrotoma suturalis
(Loew) larvae, from which my laboratory supply
derives and Richard Markley for help with the
photographs.
This investigation was supported in part by grants
to Dr. Inou6 from the National Science Foundation
(G-19487) and the National Cancer Institute, United
States Public Health Service (CA 04552). Some of
this work was carried out during the tenure of a
Predoctoral Research Fellowship from the Division
of General Medical Sciences, United States Public
Health Service.
Portions of this paper were presented to Dartmouth
College in partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
Receivedfor publication, September 9, 1964.
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endosperm, Exp. Cell Research, 25, 251.
BAJER, A., and MOL~-BAjER, J., 1963, Cine analysis
of some aspects of mitosis in endosperm, in Cine-
112
micrography in Cell Biology, (G. G. Rose, editor),
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THE JOURN2,1~ OF CELL BIOLOGY . VOLUME ~5, 1965
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COOPER, K. W., 1951, Compound sex chromosomes
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CORNUAN, I., 1944, A summary of evidence in
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APPENDIX
Focusing the Microbeam
The visible light image of the ultraviolet source
is used to focus the ultraviolet microbeam into the
specimen plane (see Zirkle, 1957). W h e n a reflecting lens is used for this focusing, the ultraviolet and
visible images should be focused to the same position. But since a refracting material is present between the lens and the focus point, namely the
quartz coverslip, light of different wavelengths
will be focused at different positions because of the
refractive index dispersion of this material. Thus,
when the visible image of the ultraviolet source is
in focus, other wavelengths are not in focus (Norris
et al., 1951; Uretz and Perry, 1957). This focus
shift is illustrated in Fig. 18 A and the calculated
focus shift for various wavelengths with respect to
the mercury green line (546 m/~) is shown in Fig.
I8 B for different thickness coverslips. (The calculation uses Snell's law, the refractive indices of
fused quartz as given in the Chemical Rubber
Publishing Company's Handbook of Chemistry
and Physics, and the application of simple trigo-
114
nometry.) The problem in focusing an ultraviolet
microbeam, then, is to correct for this focus displacement between the visible and the ultraviolet
images of the source.
Usual methods of refocusing require (a) the
ultraviolet aperture on the optic axis, and (b) motion of this aperture (or the reflecting lens) along
the optic axis for (c) a measurable distance (e.g.,
see Uretz and Perry, 1957). This situation is illustrated in Fig. 19 A: Ah is the displacement along
the optic axis (oa) between the visible (v) and the
ultraviolet (uv) images of the source (UV), which
is caused by the quartz coverslip (qz); M is the
lateral magnification and M 2 the longitudinal
magnification of the lens which is represented in
the thin lens approximation (Hall, 1953, chapter
3; Hardy and Perrin, 1932) as a straight line, L,
with focal points at F. When the ultraviolet source
aperture is moved a distance M~Ah along the axis
away from the lens to a new position (UV'), the
ultraviolet image (uv 1) for this new position is
brought into focus in the specimen plane (sp) and
the corresponding visible image (v') is in focus
TIlE JOURNAL OF CELL BEOLOGY • VOLUME ~5, 1965
A h (/z)
8 -
7 --
/k--
T =0.170
(~)--
T-0.175
•
.......
A
B
T = O,180
6 --
Z/t ,-o2,
fJ
5
ZXh
Snell's
Law
N Sin e - c o n s t a n t
Sin e =Nx2 Sin e z, -NxI Sin e I,
i
3
tan
\\~"
L~h -
I
300
I
r
8 2, - t a n Ot, ]
7o7 0
I
500
400
600
700
k (m F )
FIGURE 18
between the lens and the specimen plane. Refocusing is accomplished by moving the aperture
by M2Ah in a direction along the optic axis, or,
equivalently, by moving the lens by a distance Ah.
In Fig. 19 A the ultraviolet aperture is drawn very
large, such that the correction which focuses the
ultraviolet image in the specimen plane introduces
a lateral shift in the image position for those points
of the source which are off the optic axis (cf. uv and
u g in Fig. 19 A). Since the ultraviolet apertures
used as sources are less than a millimeter, the
lateral shift is no problem, if the apertures are
placed on the optic axis of the system.
An optical method for refocusing the microbeam was devised which eliminates the need for
accurate mechanical movement of the lens or
aperture, and eliminates the need for the ultraviolet source to be on the optic axis. In this method,
a fiat cell is inserted between the ultraviolet source
aperture and the reflecting lens while the aperture
is focused onto the specimen with visible light; the
cell is removed after focusing, prior to irradiation
with ultraviolet. The effect of inserting and removing the flat is illustrated in Fig. 19 B. Interposing
the fiat makes the actual source aperture (UV ~)
appear to come from a position, (UV), M2Ah
closer to the lens. There is no lateral shift (i.e.,
in a direction perpendicular to the optic axis) between either the two visible images (v, v ~) of the
source aperture or the two ultraviolet images
(uv, uv ~) of the source aperture when the surfaces
of the flat are perpendicular to the line between
the ultraviolet source aperture (UW) and the focal
point F, even when the source aperture is not on
the optic axis (Fig. 19 B). The flat position is
adjusted such that by inserting and removing the
flat the two visible images of the aperture occur at
the same position on an ocular grid, the visible
images being focused upon by changing the fine
adjustment of the microscope. (This adjustment
ARTHUR FORER Spindle Fiber Birefringence
115
Sp
QZ
d
4
4
--
X
013
i
V
-'"
]
4---"
v
|
UV
UV
UV
UVe
t.
B
/"~ A
$p
QZ
OCI'
UV
M~~h
r
UV i
FmURE 19
relies on the mechanical stability of the microscope fine adjustment, which, in our experience,
is quite reliable, and much better than that of the
substage lens-focusing system.) T h e fiat was a
machined cylinder of Plexiglass (Commercial
Plastics, New York) to which was cemented two
microscope slides, and into which was added
aqueous solutions of hexylene glycol. The exact
concentration varied depending on the optical
path desired. The optical path of such a flat is:
(N -q
ah'=\~-g ITs+\
(N,_q
N, ] T,
where T is thickness, and N is refractive index.
Ngl .... Tgl .... and T~olutio., were measured, and
116
N~olution was adjusted (by changing the ratios
of hexylene glycol and water) such that:
Ah' = M~Ah
where M is the lateral magnification of the reflecting lens, and Ah is the focal shift due to the quartz
coverslip as shown in Fig. 18 B. M was measured
for the aperture position in question, and Ah
was chosen for the wavelength desired, usually
275 m/~.
The advantages of the refocusing system which
used the ([at are (a) it can be used with common
laboratory microscopes, since it does not rely on
well-machined aperture movement or lens movement devices; (b) it can be used with a m i n i m u m
T H E JOURNAL OF CELL BIOLOGY " VOLUME ~5, 1965
of expensive equipment, and is as reliable as the
expensive equipment; (c) it does not require that
the ultraviolet aperture be placed on the optic
axis; and (d) it permits very quick change of the
focus correction, for no aperture position changes
need be made but only a new solution, or a new
cell, be inserted between the aperture and the re-
flecting lens. Brumberg (1943) and Norris et al.
(1951) had previously corrected for the dispersion
of the coverslip by using a substage plano-convex
lens made from the same material as the coverslip.
The optical refocusing method described here is
similar in principle to their correction, but is much
simpler in fabrication.
ARTHUR FORER Spindle Fiber Birefringenee
117