Histochemie 4, 351--356 (1965)

Department of Pathology, Government Hospital, Tel-Hashomer, Israel

T H E CAUSE OF T H E G R E E N P O L A R I Z A T I O N COLOR
OF AMYLOID S T A I N E D W I T H CONGO R E D
By
M. WOL~AN and J. J. BUBIS
With 3 Figures in the Text, of which 2 in Colour
(Received July 17, 1964)
I t has been known for almost forty years t h a t amyloid deposits stained with
Congo red exhibit a more intense birefringence than unstained deposits and t h a t
Congo red-stained amyloid appears green under the polarizing microscope (DIvR:e
and FLORKIN, 1927). MISSVlAHLand HARTWIG(1953), who studied this phenomenon,
concluded t h a t amyloid consists of ordered micelles and t h a t the apposition of
Congo red particles on the amyloid also occurs in an ordered manner. The
occurrence of the green polarization color could not be adequately explained by
these nor apparently by subsequent workers. The conclusion t h a t amyloid
consists of fibrillar elements has been proved in electron microscopic studies by
COHEN and CALKINS (1959) and confirmed by numerous other workers.
The present paper deals with some experiments and observations dealing with
the cause o/ the green polarization color o/ stained amyloid which allowed the
formulation of a consistent explanation el the phenomenon.

Experiments
1. Experiments with pure Congo red
I n one series of experiments Congo red was dissolved in 50 % ethanol and the
saturated solution was left to partly evaporate at room temperature. Smears
were made of the Congo red crystals and examined under polarized light. Fig. 1
shows that the polarization color of thick fibrils was red, of thinner was orange
and yellow and only the thinnest crystals appeared green.
I n another series of experiments a saturated solution of Congo red in 50%
ethanol was poured on a slide and left to dry. The dried surface was then polished
in one direction b y pressing it against a turning glass wheel of a microtome knife
sharpener. Examination of the polished smear for dichroism revealed that some
dichroism could be detected in all areas. The color of the dye was reddest when
the direction of the polish and t h a t of the polarizer coincided. However, a com-
plete transition of red to colorless or almost colorless occurred only in those
areas where the smear was thinnest. Examination of the various areas for the
color of birefringence revealed that only in those areas where dichroism was most
pronounced (colorless to red) did the green polarization color appear under
crossed polars. In thicker areas of the smears (where the dichroitic difference was
from red to darker red) yellow, orange and red polarization color was noted under
crossed polars (Fig. 2).
Histochemie, Bd. 4 25
352 M. WOMANand J. J. BUBIS:

F i g . 1. C o n g o r e d c r y s t a l s e x a m i n e d u n d e r p o l a r i z e d l i g h t . C h a n g e i n c o l o r f r o m r e d t h r o u g h y e l l o w
t o g r e e n i n a c c o r d a n c e w i t h t h i c k n e s s of t h e c r y s t a l s . 300

a t b --~ c

F i g . 2a---c. D r i e d s m e a r o f C o n g o r e d p o l i s h e d w i t h a g l a s s w h e e l . I n a t h e a p p e a r a n c e of t h e f i e l d
w i t h t h e l e n g t h of t h e s m e a r p a r a l l e l t o t h e l i n e of p o l a r i z a t i o n o f t h e p o l a r i z e r . N o t e t h e r e d a p p e a r -
a n c e o f t h e f i b r i l s . I n b t h e s a m e f i e l d w i t h t h e l e n g t h p e r p e n d i c u l a r t o t h e p l a n e of t h e p o l a r i z e r .
Note t h a t some areas which a p p e a r e d red i n a are colorless here. I n c t h e appearance u n d e r crossed
polars. Note t h a t only t h e areas which were p r a c t i c a l l y colorless in b are green here. The o t h e r areas
a r e y e l l o w t o r e d . 30

2. Optical e//ects o] Congo red staining on various tissues

A number of operating theatre biopsies which contained different types of
hyalinized areas and some amyloid-containing organs, were fixed in 10 % formalin
or in a modified Stieve solution (containing 1% HgC12) and were embedded in
paraffin and sectioned at 6--8 microns. I n some instances marked sites in the
sections were examined for intensity of birefringence (with the Berek compen-
sator) before and after staining with Congo red. I n other instances two con-
tiguous sections were examined, the one unstained but completely dewaxed
overnight in xylol and the other stained with Congo red.
 Green polarization color of Congo red-stained amyloid 353

Sections to be stained with Congo red were immersed in xylol overnight to

ensure adequate deparaffination, blotted and hydrated (if necessary, after treat-
ment with Lugol's solution). They were then stained for 5 minutes in a 0.5%
solution of Congo red in 50% ethanol, blotted, differentiated for 15--30 seconds
(until no more stain could be seen leaving the section) in a 0.2% solution of K O H
in 80% ethanol, rinsed for one minute in running water, counterstained with
Mayer's hemalum, dehydrated and mounted in Canada balsam.
The various tissues stained with Congo red were examined for the occurrence
of dichroism by using only the polarizer of the polarizing microscope without
the analyzer. The effect of the staining on the intensity of birefringence was also
studied. The equipment used consisted of a polarizing Leitz microscope with a
Brace-KShler and occasionally also the Berek polarization compensators.
I t has been found t h a t dichroism was marked in amyloid and especially in
thin amyloid fibers. No obvious diehroism could be detected in elastic or
collagen fibers or in masses of hyalin which stained with Congo red.
I n accordance with known facts staining with Congo-red markedly increased
the intensity of birefringence of amyloid. The effect of Congo red-staining on the
other tissues was variable: in most instances the intensity of birefringence re-
mained practically unchanged. In other materials there was either a slight
increase or a slight decrease.

3. E//ect o/thickness o/the amyloid deposits

The effect of increasing the thickness of amyloid-containing tissue sections
subsequently stained by Congo red has been studied by DIEZEL and PFLEIDERER
(1959) and can be readily confirmed: with increasing thickness of sections the
Congo red-stained amyloid appears yellow, orange and finally red under crossed
polars.
In the presently described experiments blocks of amyloidotic kidney and
adrenal which were fixed in formalin and embedded in paraffin were cut at
1--2 micra. The sections were then stained with Congo red as above. The
weakly birefringent amyloid deposits appeared faint reddish under crossed polars.

Discussion
The experiments done on Congo red crystals indicate that the green polariza-
tion color depends, at least in part, on the thickness of the object. Clear-cut
results were obtained with the polished smear. The procedure of polishing dry
smears of dyes in order to obtain orderly arrangement of micelles and thereby
induce intense and clear dichroism has been proposed and used by ZOCHER and
JACOBu (1927). In such smears it became obvious that a perfect arrangement of
the dye molecules (which is shown by a "clean" red to colorless dichroism) is a
prerequisite for the occurrence of green polarization color. This conclusion is
based on the fact that Congo-red is known to show dichroism of red, when the
micelles are arranged parallel to the direction of the polarizer, to colorless, when
they are perpendicular to the polarizer (ScHMIDT 1956). Yellow and pink color of
material examined with only the polarizer in the perpendicular direction indicate
that the molecules are not perfectly aligned and the resulting color is a mixture
of red and colorless.
25*
354 M. WoLmr and J. J. BUBIS:

The study of sections of amyloid-containing tissues stained with Congo red

indicates that the occurrence of green polarization color also depends on thickness
of the object. Green color appears only within a certain range of thickness, while
1--2 microns thick sections as well as those 20 microns thick do not show the
phenomenon.
The findings can be explained as follows. I n a perfect crystal of Congo red, in
which all the molecules lie parallel, examination for dichroism will show transition
from red to colorless when the direction
of the crystal varies from that of the
polarizer's axis to a position perpen-
dicular to it. This phenomenon depends
on the degree of "order" i.e. parallelism
in the arrangement of the molecules
+I/"
and not on the thickness of the sample.
A k'_.,.~, o When a near-perfect crystal of Congo
o
+
red of a given thickness is examined
under crossed polars when situated at
450 to the polarizer axis, the polarized
light which enters the object (Fig. 3) is
split into two rays, a red one which runs
parallel to the length of the crystal and
a white one perpendicular to it. The red
component of the white ray is obviously
of the same amplitude (and therefore
intensity) as the red ray. On being
brought together by the analyzer the
P
~ two rays can produce phenomena of
interference. Whenever the retardation
of the slow ray is equal to half the
wavelength (6) of red fight, the red ray
12ed
. . . . . Wh//-e
.
will cause complete extinction of the
Fig. 3. S c h e m a t i c d r a w i n g e x p l a i n i n g t h e pro-
duction of g r e e n polarization color b y Congo-red-
red component of the white ray. Partial
s t a i n e d amyloid. P polarizer; A m a m y l o i d ; extinction of nearby wavelengths will
A a n a l y z e r . + the r a y is t r a n s m i t t e d ; o r a y is
not transmitted also occur, and this will be more
accentuated in one or the other direction
of the spectrum when the thickness of the object will be slightly more or less
than t h a t which is necessary for a ~ retardation. Extinction of red produces
the appearance of the complementary c o l o r , - green.
I t is clear t h a t two factors are needed for the production of the green polariza-
tion color :1) a near-perfect parallel alignment of the molecules of Congo red on the
one hand, and 2) an adequate thickness of the object to produce a 2 retardation.
Amyloid appears to differ from other structures and materials which are
stained by Congo red in one characteristic. Staining of amyloid with Congo red
(and possibly also with other dichroitic dyes) is associated with a degree of order
in the arrangement of the dye molecules which is not present in any other material.
The perfect alignment might indicate a stronger dipole moment in the molecules
of amyloid than in the other molecules, or else the presence of channels within
 Green polarization color of Congo red-stained amyloid 355
b

amyloid fibrils which do not allow any tilting (a possibility which might find a
morphological counterpart in the electronmicroscopical study of GUEFT and
GmDONI (1963). The greater amount of order and parallelism between molecules
of Congo red in amyloid as compared to hyalin and elastic materials is also con-
firmed by the finding that only in amyloidosis is the intensity of birefringence
markedly increased by staining with Congo red. The increased birefringence
caused by staining with Congo red is caused by the fact that Congo red molecules,
which are positively birefringent and dichroitic (W;~LCHLI, 1945), are arranged
parallel to the length of the amyloid fibrils.
It is clear that only amyloid deposits which will produce retardation of
approximately half a wavelength of red light will appear green under crossed
polars.
The general acceptance of the green polarization color as a criterion for
recognizing amyloid is due to the fact that nearly all laboratories use sections
which are 5--10 microns thick in routine work. With the thinner sections amyloid
will appear a somewhat bluer green and with thicker ones a more yellowish green
and only thick sections which are unsuitable for adequate microscopic study will
not show the phenomenon.
The retardation of half a wavelength of Congo red-stained amyloid represents
the sum of two factors: the intensity of birefringence of unstained amyloid
multiplied by the thickness of the deposit plus the intensity of birefringence of the
ordered Congo red multiplied by the thickness. If the thickness of the Congo
red-stained deposit is t, the intensity of birefringence of unstained amyloid n a
and the intensity of birefringence of the ordered Congo red n o , then the retarda-
tion r will be : r ~ t . n a - ~ t" n c - - t ( n a ~ n c ) .
I t is clear from this formula (and from the result of the previously described
experiment with a thin section of amyloid) that very small (or rather thin)
amyloid deposits will escape detection between crossed polars. The intensity of
staining with Congo red of such deposits is so low that they will certainly also
escape detection with ordinary light. It is suggested that the best procedure for
detecting such minimal deposits of amyloid consists of examining for dichroism
(i.e. only with the polarizer) under ultraviolet light sections stained by fluorescent
dyes. By this means the dark field effect will also be obtained in the study of
dichroism. Fibrils which will not appear in the one direction and which will be
fluorescent in a perpendicular direction, will be assumed to represent amyloid
even if they do not show the green polarization color in Congo red-stained
sections.
Summary
Experiments done with Congo red crystals and with Congo red deposits polished
in a single direction by a glass wheel have shown that the appearance of green
polarization color primarily depends on near-perfect parallel alignment of the
dye particles. The green polarization color was seen only in the deposits which
showed a clear transition from red to colorless when examined for dichroism.
Another factor was found to be the thickness of the object, as the green polariza-
tion color was not present in too thick or too thin sections of amyloid-containing
tissues stained with Congo red.
356 M. WOLMANand J. J. BUBIS: Green polarization color of Congo red-stained amyloid

The p h e n o m e n a can be e x p l a i n e d b y t h e a s s u m p t i o n t h a t t h e green polariza-

tion color is due to interference b e t w e e n t h e r e d r a y a n d t h e r e d c o m p o n e n t of t h e
white r a y w h e n e v e r t h e r e t a r d a t i o n b y t h e o b j e c t a p p r o x i m a t e s half t h e wave-
length of r e d light.
The findings i n d i c a t e t h a t a m y l o i d differs from o t h e r m a t e r i a l s which arc
s t a i n e d b y Congo r e d in t h a t a m y l o i d deposits b i n d the d y e molecules
in a m o r e o r d e r l y a n d parallel fashion. I t is suggested t h a t m i n i m a l a m o u n t s of
a m y l o i d which are n o t visible in Congo r e d s t a i n e d sections with o r d i n a r y light
m i c r o s c o p y a n d which do n o t give t h e green p o l a r i z a t i o n color can best be
d e t e c t e d b y e x a m i n a t i o n for dichroism in u l t r a v i o l e t light after h a v i n g been
s t a i n e d with fluorescent dyes.

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Prof. M. WOLMAN,
Government Hospital, Dept. of Pathology, Tel-Hashomer/Israel