Journal
of The
Franklin Institute
Vol.237
FEBRUARY, 1944 NO.2
THE
NEW MICROSCOPES.
A
Discussion by
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1
R.
E. SEIDEL, M.D. AND M. ELIZABET WINTER,
PhiIadelphia , Pa.
It is, to
speak conservatively, of extreme interest to review the recent
progress made by the scientist in his endeavor to penetrate the
unseen world of the minute and disease-causing organisms, in
particular a world of viruses-suspected; yet lying just beyond
the scope of human vision and the power of the microscope to
reveal; for the laboratory research worker, the doctor, the
technician long have been familiar With the effects of these
unseen enemies they have been called upon to treat and to cope
with in man, animal, and plant, and while their knowledge of the
infinitesimal has been growing steadily, they were, until very
recently,' unable to make the slight step "beyond"
which would enable them to "see." But today, Science
is exploring looking for the first time upon totally new worlds
through the eyes of totally new types of microscopes,
microscopes new in principle of construction and in principle of
illumination.
THE
ELECTRON MICROSCOPE.
One
of these new instruments the Electron microscope, has received
considerable attention and is now being used extensively in both
industrial and medical research. Based on the principles of
geometric electron optics, this microscope utilizes electrons as
a source of illumination instead of the light source of the
ordinary light microscope.
Electrons,
practically speaking, are the smallest, lightest particles of
matter and electricity. Like light, they behave like corpuscles
guided by waves. Unlike light, however, they travel in a
straight line in a vacuum where, subject to the action of
electric and magnetic fields, their behavior coincides with the
laws and principles set down by Sir William Hamilton who, more
than a century ago, demonstrated the existence of a close
analogy between the path of a light ray through refracting media
and that of a particle through conservative fields of force.
We know
that these negatively-charged particles, the electrons,
revolving about in their various orbits in the atom, serve to
maintain the balance of the atom while the nucleus exerts the
"positive" force which holds it together; and we also
know that when this balance is upset, due to gain or loss of
electrons, we think of the atom as "charged," since it
is this circumstance which causes the tiny particle to attract
or
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2
repel
other electrons according to the state of its unbalance. And
Science has succeeded in unbalancing the atoms to such an
appreciable extent that the negative electricity may be
withdrawn and harnessed for use in such instruments as the
Electron Microscopes.
The fact
has long been established that atoms are in a constant state of
vibration in a heated body and that the greater the heat of the
body, the greater the agitation of the atoms. According to the
electron theory of metals, electrons circulate about a
three-dimensional network, or lattice, of positive ions1 some of
the electrons being comparatively free, that is to say, the
attractions of the ions are practically cancelled by the
repulsion's of the other electrons. It does not necessarily
follow, however, that the same electrons consistently remain
free. They may be controlled by the ions eventually, but
regardless of this, there is always a fixed number of them that
are free. Moreover, there is a critical value of speed above
'which the electrons are able to rise in metals and thus escape
from their restraining positive charges, though at ordinary
temperatures the proportion of them moving rapidly enough to do
this is relatively small. However, as the heat applied to the
metal is increased, not only is the thermal agitation of the
electrons increased also, but the proportion among them
possessing sufficiently high speeds to enable them to leave the
metal.
Thus
is heat applied to the electron source of the Electron
Micro-scope which, in the case of most instruments of this kind,
is a tungsten filament surrounded by a guard cylinder. After
leaving the filament, or cathode, the electrons enter an
electric field wherein are large accumulations of charge which
serve to steadily speed up the motion of these freely-moving
particles. Since the electrons travel in vacuum, none of the
kinetic energy gained in crossing the field is lost, the total
kinetic energy, or energy of motion, gained in passing through
this region being proportional to the voltage applied. We may
deduce, therefore, that since increase of charge in an electric
field means a proportional increase of kinetic energy of these
electrons, the higher the voltage applied? the greater the speed
of the electrons-all of which has been calculated mathematically
and confirmed experimentally.
After
traversing the electric field and passing through the anode, the
electrons are concentrated on the specimen under examination by
the first of three magnetic fields which are created by currents
flowing through coils enclosed in soft iron shields, molded so
as to concentrate the magnetic fields on a short section of the
microscope's axis. Whereas in the ordinary light microscope
glass lenses serve as the refractive media through which light
rays are deflected, in the Electron Microscope it is these
magnetic fields of rotational symmetry which are the refractive
media and serve as the "lenses" which deflect the
beams of electrons. The first of these, the condenser lens coil,
corresponding to the substage condenser of the ordinary light
microscope concentrates
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the
beam of electrons upon the specimen. The convergence of the beam
falling on the specimen is controlled by varying the current
through this condenser lens. Now, having passed through the
specimen, the objective coil, similar in effect to the objective
lens, focuses the electrons, and an intermediate image enlarged
about one hundred diameters is formed. Finally, the projection
coil corresponding to the projection lens or ocular, produces a
further magnified image on a large fluorescent screen. In some
of the Electron Microscopes, there is a periscope-like
attachment by means of which it is possible to locate and adjust
for study the most interesting portion of the specimen. or that
which it is desired should be examined, before the projection
lens coil forms the final magnified image upon the screen, since
it is sometimes difficult to accomplish this at high
magnification. Also, if it is desired that a photographic record
be made, the screen can be removed and a photographic plate
substituted.
The
specimen itself is supported on a thin nitrocellulose membrane
less than one-millionth of an inch thick, and clamped in the tip
of a cartridge which is inserted between the pole pieces of the,
objective coil. The membrane is suspended across the opening of
a fine mesh screen, and a plate, serving as the movable stage,
supports the cartridge. The image is projected onto the screen
according to the density and atomic weight of the specimen. In
other words, whereas in the ordinary light microscope the image
is seen due to refraction of the specimen or differences in
absorption, in the Electron Microscope the image is seen due to
scattering of the electrons, and since electrons travel in a
straight line in a vacuum, it stands to reason that even a
fairly thin specimen will prove sufficient to deflect such
particles. Electrons which strike a thick or solid portion of
the specimen will, of course, not continue on in a straight line
to the screen but will be either completely absorbed by the
specimen or scattered too far out of the beam, thus failing to
enter the narrow aperture of the objective, so that that portion
of the screen corresponding to the thick portion of the specimen
will remain dark. However, those electrons which are able to
escape complete absorption or too great deflection because they
do not happen to come in contact with too solid a portion of the
specimen and either pass along on all sides of it or penetrate
the thinner portions where it is possible they may encounter
only a single heavy nucleus for considerable scattering (the
angle of deflection being proportional to the square root of the
thickness), continue on to the screen where they impinge and
cause the, chemically-treated screen to fluoresce, thus
providing a study in light and shadow. If the atoms of a
particular substance are heavy, they will also deflect more
electrons than if they were light. It may be readily seen,
therefore, that the thinner the specimen and its mounting, or
the greater the variations in density of the specimen, the more
internal structure and detail which may be seen, since too great
density
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tends
to absorb or interrupt the straight-line progress of too many of
the electrons.
Focusing
of the image is accomplished by varying the strength of the
fields and thereby altering the focal length of the
"lens" coils at will1 so that the need of changing the
specimen 5 position in relation to a fixed optical system as
would be the case with and ordinary light microscope, is
avoided. Thus, magnification in an Electron Microscope can be
continuously varied.
Some
specimens may be mounted directly on the fine mesh screen while
others may be embedded in collodion, sealed between films of
collodion, or suspended in a gelatin film, itself supported on
collodion film. The supporting films beside being very thin must
be homogeneous lest an artefact be created. For the most part,
no staining of bacteriological specimens is done since usually
they exhibit sufficiently high contrast in density to readily
reveal flagella and other detail without any preparation except
that of suspending the specimen in distilled water or other
liquid and allowing a drop of the suspension to dry on the film
surface which method is also utilized for specimens of colloidal
particles, pigments, and other chemical preparations. At times,
however, as Dr. L. Marton of Stanford University has mentioned
in his article on the Electron Microscope (written for The
Journal of Bacteriology, March 1941, when he was associated with
the R. C. A. Research Laboratories), virus particles may show
decided low contrast. One method which Dr. Marton mentioned for
overcoming this is to secure a number of electron micrographs at
various focuses and simply select the best one for study. Or the
virus may be permitted to absorb colloidal gold which would
result in an image of high contrast. Dr. Marton points out that
there may be future need for a staining in density and that
already osmic acid has been tried and used for this purpose.
In
this microscope, voltages of between 30,000 and 60,000 are used.
It has been previously stated that the higher the voltage, the
greater the speed of the electrons. This might now be augmented
to the higher the voltage, the greater the speed of electrons;
hence, the shorter the wavelength. An explanation of this may be
approached through a brief discussion of short-wave diffraction
as considered Dr. Karl K. Darrow of Bell Laboratories in
his book, "The Renaissance of Physics." In order to
obtain convenient angles of refraction with the ordinary
diffraction grating, it is necessary that the wavelengths of
light be smaller, but not many times smaller, than the spacing
between the wires or grooves. Naturally, a limit of measurement
is reached in the region of ultra-violet light since it is
impossible to further lessen the spacing of these gratings.
However, this limitation was overcome when von Laue conceived
the idea of substituting a crystal for an artificial grating
since the atoms in a crystal are a thousand
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times
more closely set together than are the wires or grooves of a
grating and are arranged in precise regular order or
"lattices," and, like gratings, are unable to diffract
waves which are longer than the spacings between their atoms.
Von Laue suggested that if a beam of light were directed across
a crystal and made to strike a photographic plate, there would
appear a spray of narrow rays each composed of a single wave
train instead of the broad fan-like. arrangement of the grating,
and a pattern of star-like spots where the rays come in contact
with the plate instead of the dark irregular blot when a grating
is used. Of course the rays are disposed according to the
spacings of the atoms in the lattice and according to the
character of the lattice. Von Laue confirmed this idea for waves
short enough to be so diffracted and then advanced the theory
that this principle might hold true for x-rays as well, which
theory was almost immediately confirmed by Friedrich and
Knipping. Shortly after Schroedinger began to develop De
Broglie's wave theory of electrons, Elsasser conceived the idea
that possibly these tiny particles might also be diffracted by
crystals, and Doctors Davisson and Germer of the Bell Telephone
Research Laboratories, using as part of their apparatus an
electron gun, set out to test and to prove this theory Due to
their experiments and those of G. P. Thomson, it was established
beyond a doubt that electron beams are diffracted just as are
x-ray beams. However, it was also demonstrated in the course of
these experiments that electrons of slow speeds and feeble
kinetic energies are unable to penetrate the crystals. It was
Thomson who utilized faster electrons and demonstrated that not
only are electrons diffracted like x-rays, but like x-rays also
they make an imprint upon a photographic plate at increased
speeds. These three men, together with others, then measured the
wavelengths which they compared with the momenta of these
electrons by their diffraction. To these experiments and
measurements were then applied the following Rules of
Correlation:
"Energy
(E) is proportional to frequency (v), and momentum (p) is
inversely proportional to wavelength (lambda), the same constant
(h) appearing in both relations. (Frequency is interpreted as
the velocity (V) of the waves divided by their
wavelength.)" These Rules can be applied mathematically to
the Electron Microscope to better illustrate the principles of
its operation. In making use of the first Rule, however, it is
necessary to substitute "voltage" for
"frequency," and in so doing, therefore, the Rules of
Correlation explain the increase of energy in relation to the
increase of voltage as well as the increase of speed of
electrons in relation to the decrease or shortening of
wavelength when we say-the higher the voltage, the greater the
speed; hence, the shorter the wavelength of electrons. It is
interesting to note in passing that a 150-volt electron has a
wavelength of one angstrom unit, this being more than 10 -3
times smaller than the wavelength of visible or ultra-violet
light.
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Because
the wavelengths utilized in an Electron Microscope are so much
shorter than those employed in an ordinary light microscope, it
is possible to obtain greatly increased resolution and
magnification. As a matter of fact, resolution up to 20,000 or
25,000 diameters may. be realized, and increased magnifications
beyond this point up to 100,000, even 200,000 diameters, can be
obtained, such magnifications, however, constituting enlargement
of the image. (Definitions of "Resolution" and
"Magnification" discussed under "The Ordinary
Microscope") This high magnification is greatly desirable
since otherwise the eye would be unable to distinguish the fine
detail of internal structure at a resolution of the order of
25,000. As a result of this 'increase in resolution and
magnification over that of the ordinary light microscope which
is between 1,6oo and 2,500 diameters and in the ultra microscope
between 2,500 and 5,000 diameters, many surface cells and much
intricate internal structure hitherto unsuspected, or at least
undetected by ordinary microscopes, have been revealed. To cite
a few examples:
The
streptococcal cells appear, not as individual cells, that is,
separate and apart from one another, but as chain-like groups,
the cells in each chain being bound together apparently by the
strong rigid membrane or outer cellular wall which extends over
a number of these cells and which is so plainly evident under
the Electron Microscope. Subjected to sonic vibration, these
cells suffer a loss of protoplasmic material from their
interior, causing them to become mere "ghost" cells,
which makes them more transparent to electron beams. That there
exists considerable difference. between the surface structure
and internal composition of these cells has also been determined
and (demonstrated.
Using
the Electron Microscope, Dr. Harry E. Morton of the Department
of Bacteriology of the University of Pennsylvania Medical School
and Dr. Thomas F. Anderson of R. C. A. Research Laboratories
were able to demonstrate that in at least one instance where
chemical reaction is induced by bacteria this reaction takes
place "inside" the cells. The fact that diphtheria
bacilli reduce potassium tellurite to metallic tellurium has
been known for some time, but whether this reaction occurred
inside the cell or on the cell surface or both had never been
definitely shown until the Electron Microscope was made
available. Then, securing unstained preparations of
Corynebacterium diphtheriae grown on blood infusion agar, Drs.
Morton and Anderson demonstrated that the typical polar granules
appear as dense spherical masses, or possibly plates, of a very
black color and that in unstained preparations of this same
Corynebactenum diphtheriae grown on potassium tellurite
chocolate agar, not only the polar granules are in evidence but
also the tiny needle-like crystals inside the cell which
disappear along with the black color of the cell masses when a
drop of bromine water is added to I cc. of a suspension of the
cells on potassium tellurite chocolate agar.
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From
this the experimenters were able to deduce that tellurium metal
occurs in the form of needles and is the cause of the black
color, and that this reaction occurs within the cells since the
crystals have never been observed to lie totally outside the
cell wall, although at times there is some distortion of the
wall.
The
Electron Microscope also affords such study and Observation as
that carried out by Dr. \V. M. Stanley of the Rockefeller
Institute for Medical Research and Dr. Thomas F. Anderson in
their recent investigation of plant viruses. By means of
electronmicrographs, they were able to judge the exact manner
and extent of attack made on the tobacco mosaic virus by the
protein antibodies in the blood stream of rabbits in which an
artificial immunity to the virus had been produced.
Structures
like that of the spirochete of Weil's disease, typhoid flagella,
unusual internal, structure of pertussis organisms, tubercle
bacilli, the isolation and recognition of the influenza virus,
the spores of trychophyton mentagrophytes, spirochaeta pallida
with its accompanying flaggelar appendages, and colloidal
particles are but a few of the interesting revelations of the
Electron Microscope for medical science.In-dustrial science,
too, has found this new research tool of great value in the
study of metals, alloys, and plastics, as well as in the study
of size, shape,and distribution of particles in chemical
compounds and elements.
The
Electron Microscope herein described is that manufactured by the
Radio Corporation of America. There are, of course, variations
in construction of the different instruments of this kind but
all types are built along similar lines and upon the same
general principles. In the Electron Microscope there is some
aberration plus the additional disadvantages of having the
specimen in a vacuum, not to mention the probable protoplasmic
changes induced by the terrific bombardment of electrons, and
finally, what is perhaps the greatest disadvantage insofar as
medical science is oncerned-that of being unable to view living
organisms. Nevertheless,thedisadvantages of the microscope are
far overshadowed by its increased resolving and magnification
powers which have combined to make it an invaluable research
tool.
RESOLUTION
AND MAGNIFICATION OF ORDINARY MICROSCOPE.
We
have stated that the resolving power of the ordinary light
microscope is restricted to between 1,600 and 2,500 diameters
and that of the Ordinary ultra microscope to between 2,500 and
5,000 diameters, resolution in any microscope being the ability
of the instrument to reveal the most minute of component parts
of a specimen so that each may be seen as a distinct and
separate image. For instance, let us suppose an object is
examined through which run two very fine parallel lines closely
set together. If the two lines are visible under the microscope
and are revealed as two separate images, then, appar-
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ently, no limit
of resolution has been reached; but if the two lines are merged
or revealed as only one, and upon further magnification the
image merely becomes enlarged without separation of the lines,
then a limit of resolution apparently has been reached and
additional magnifi-cation would constitute only enlargement.
Assuming now that the object is a point object in which case the
images of the points would be diffraction disks, the disks
should likewise be sufficiently resolved so that each may be
distinguished as a single image. If, when these disks are seen
to overlap, additional magnification fails to extend the
distance between them, their size simply increasing in
proportion to the increase of magnification, or, if they are all
but completely merged and the image becomes just a spurious disk
of light, it is evident that a definite limit of resolution has
been attained and that further magnification would be useless.
Resolution, in a broad sense, then, is the ability of the
microscope to bring out or reveal internal structure and detail
of a specimen, the shortest distance it is possible to separate
two component parts, according to Abbe, being not less than the
wavelength of light by which the specimen is illuminated divided
by the numerical aperture of the objective lens plus the
numerical aperture of the condenser lens, or, about one-third
the wavelength of light utilized.
The
several factors which are generally acknowledged to be
responsible for the limitation of resolving power are
inter-related. Now when light passes from one medium into
another of different density, in the instance which we are
considering that of light refracted by the specimen and passing
from air into glass, the light rays are deviated from their
straight-line course; that is to say, that when they come to
within a very short distance of this denser medium, they are
acted upon by a very powerful force in such a manner that they
execute a short rapidly curving motion, or an angle, and are
pulled into the medium of greater density. When the rays of
light undergo such a force, the momentum of the corpuscles is
increased and the speed of the waves (decreased, resulting, of
course, in a shortening of the wavelengths. Here, again, we may
make use of the second of the Rules of Correlation Momentum (of
corpuscles) varies inversely as wavelength (of waves)."
Once well inside the new medium, however, the light rays
straighten themselves out again (unless the medium is so
constructed that it possesses gradation of density in which case
they follow a curved path). They do this in spite of the fact
that the same forces are still acting upon them, although now
these forces issue from all sides of them and so cancel each
other out, the momentum of the photons or light corpuscles
continuing to increase while the speed of the waves is
proportionatelv retarded. If the light is refracted normally to
the surface, however, it does not bend, but tends to cause a
shortening of the optical path although the wavelength is
shortened regardless. It is only when it is refracted obliquely
to the surface that the light is bent, the greater
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being
the obliquity of the incident ray and the denser the medium, the
greater the bending of the angle of the cone of light and the
shorter the wavelength. It might therefore seem desirable to
obtain as great an angle of refraction as possible. However,
shortening of the wave-length is not in exact proportion to the
amount of bending except in the case of the diffraction grating.
And regardless of how great a change there is in its angle, the
numerical aperture of the light, or angular aperture as it is
more properly called, remains constant.
In
order, then, that the cone of light be large enough to supply
the aperture of the objective with sufficient light to produce
an accurate, bright, and enlarged image of the specimen, it is
first necessary that the specimen be refracting or emitting
light of an adequate quantity. since both magnification and
resolution are largely dependent upon the amount of light which
the objective utilizes and receives into the tube of the
microscope and since such' light as the objective does receive
should be only that emitted by the specimen. It is obvious,
therefore, that it is of primary importance for the specimen
itself to be amply illuminated. This would seem to depend
entirely on the actual light source, yet no matter how powerful
a light source is employed, it is of little avail unless the
condenser is of sufficient quality and aperture dimensions to
accommodate the light which it receives from the source. If, for
instance, the numerical aperture of the objective is 1.25, the
width of the cone of light emanating from the specimen should
completely fill this aperture in order for the fullest powers of
the microscope to be realized. Now, since the condenser supplies
the light to the specimen, it stands to reason that it, also,
should ha'-e a numerical aperture of at least 1.25. However, if
the condenser and specimen slide are separated by air, the
condenser can provide light of only 1.00 N. A. to the specimen
since, according to a law of optics, no aperture greater than
1.00 N. A., (this being the refractive index of air), can pass
from a denser medium into air. To remedy this situation, an
immersion fluid is placed between the top of the condenser and
the lower side of the specimen slide as well as between the
specimen and the objective lens.
Since
no optical medium has an index of refraction greater than three
and no immersion fluid an index of refraction greater than 1.7,
to further increase resolving power, then, might it not be
feasible to widen the apertures of the objective and condenser
lenses, thus afford mg additional illumination for utilization
by both specimen and objective? This idea would be entirely
practical except for the fact that such enlargement of the
lenses would increase aberration, both spherical and chromatic,
and apparently present-day lenses are now as highly corrected as
it is possible for human ingenuity and skillful workmanship) to
make them. Spherical aberration, caused by the paraxial rays
coming to a focus at the center of the lens before those rays
near the
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principal
axis, is corrected by using concave and convex lenses of
different material and, consequently, of different refractive
index. In this manner spherical aberration of a convex lens, for
instance, can be overcome, without its converging action being
altered, by adding to the optical system a concave lens in which
there is an equal and opposite aberration. Chromatic aberration,
occurring when more than one wavelength of light is used to
illuminate the specimen, is due to the fact that the shortest
waves of the spectrum are refracted most and the longest waves
least, thus causing the blue-violet waves to come to a focus
ahead of the red waves and resulting in a series of colored
fociall along the axis. Now since, as we have said, the
shortening of the different groups of wavelengths is not in
exact proportion to their bending and since this circumstance
varies according to the substance the light rays pass through,
it is possible to combine lenses or lens systems in such a way
that white light may be obtained. For instance, a small concave
flint-glass prism produces the same amount of dispersion as a
large convex crown-glass prism. Thus, if these two prisms are
placed with their edges opposite, the crown glass will bring
together the spectrum produced by the flint glass and white
light will be the result. However, the rays of white light will
not extend parallel with the original direction but will bend
toward the base of the crown glass since the mean refraction of
the crown glass is greater than that of the flint glass.
Achromatic objectives, corrected spherically for one color,
chromatically for two; semi-apochromatic objectives, possessing
moderate refractive indices and very small dispersion, in which
a lens of fluorite is substituted for one of the glass lenses;
apochromatic objectives, corrected spherically for two colors,
chromatically for three; and also certain monochromatic lenses
for use with light of one wavelength only are available for
overcoming, at least in part, one of the conditions which tends
to interfere with better resolution. Condensers, also, can be
corrected for both spherical and chromatic aberration and must
be achromatic-aplanatic if the light which enters the objective
is to come only from the specimen, for condensers with spherical
and chromatic aberration are unable to direct their entire cone
of light upon the specimen.
In
addition to being as highly corrected as possible and possessing
a large numerical aperture, an objective should also be capable
of adequately magnifying the image, being aided in this by the
ocular which also serves at times to compensate for the defects
in chromatic magnification which cannot be managed conveniently
by high-power objectives, the magnification of the final image
being the product of the magnification of the objective
multiplied by the magnification of the ocular. An amplifier is
sometimes inserted between the objective and ocular which causes
the rays of light from the objective to diverge to a greater
extent, thus doubling the size of the image. Magnification may
also
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be
improved by increasing the tube length, by increasing the
distance from which the image is projected, and by altering the
positions of
various
lenses in an adjustable objective. In general, greater the
magnification, the smaller will be the specimen field, but, as
has been stressed, high powers of magnification should always be
accompanied by equally high powers of resolution.
As
we have seen, resolution in the ordinary light microscope is
definitely restricted by a number of inter-related elements.
Even when monochromatic light is employed, there is always
present some spherical aberration with which to contend. True,
better visibility of specimens is provided by dark-field
microscopy in which the specimen is viewed by the high contrast
of its own scattered or reflected light against a dark field,
although in this type of illumination objects in the field must
be well separated. Much fine detail and brilliant color of
specimens can be observed by means of the polarization of light.
Further, it is possible to illuminate the specimen with shorter
and shorter wave-lengths of light, the shorter the wavelength of
light used, the more of the fine detail of the specimen which
can be seen, but a limit is reached here, also, for ordinary
glass lenses are not transparent to ultra-violet rays. However,
in the ultra-violet microscope, having a resolution twice that
of the instruments using ''visible light,'' the condenser,
objective, and ocular are all made of quartz and, by
substituting the photographic plate for direct observation, many
excellent micrographs of numerous varieties of organisms and
cellular structures can be made. But when viewed directly,
nothing of the nature or structure of the specimen can be
ascertained; only the light scattered by the specimen is
distinguishable, the size of the specimen being roughly
estimated by the amount of light refracted.
These
seemingly unsurmountable obstacles of the ordinary micro-scopes
would appear to indicate that Abbe's law and the contention of
physicists that "any object which is smaller than one-half
the wave-length of light by which it is illuminated cannot be
seen in its true form or detail" are destined to remain
undefied.
REDUCTION
IN THEORETICAL LIMIT OF RESOLUTION DEMONSTRATED.
But
Dr. Francis F. Lucas of the Bell Telephone Research Laboratories
and Doctors Louis Caryl Graton and E. C. Dane, Jr., of the
Department of Geology, Harvard University, have very
convincingly demonstrated a reduction in these theoretical
limits of resolution and visibility with their instruments,
designed for use in the visible light region of the spectrum.
The
Graton-Dane microscope is mounted on a 360 kg. steel foundation
bed which, in turn, is supported by six rubber-in-sheer
marine-engine mountings-this for the purpose of eliminating all
vibration and insuring stability of parts, two factors upon
which both men have laid
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great
stress. . Any type Source, such as the carbon arc, metallic arc,
incandescent filament, Point-O-Lite, Mercury Vapor, or any of
the special forms of monochromators, can be used for
illuminating the specimen with direct and dark-field
transmitted, vertical and oblique reflected, or polarized light.
The image. beam itself follows a straight-line path in passing
from the objective, the objective ranging anywhere from the
shortest to the greatest in. working distance, through the tube
to the ocular, as few lenses as possible being placed in its
way. The spiral-cut rack and pinion which moves the stage and
sub-stage assembly in longitudinal tracks or guides can be
operated by hand or by an electric motor and is independent. of
the fine adjustment, also motordriven, which moves only the
objective and the carriage carrying the objective. Whereas
manual operation of the fine adjustment which is one hundred
times more sensitive than that of the ordinary instruments
necessitates five hundred turns of the knob to move the
objective a distance of but one millimeter, (an adjustment
calculated to require a time period of twenty-five minutes), by
means of the motor it is possible to move the objective at the
rate of 0.01 mm. per second or 0.004 mm. per second, depending
upon which of the two speeds is desired1 rapid motion being used
when the image appears considerably out of focus and decreased
speed being used when the image seems to be reaching a point of
perfection.
Resolution
up to 6,ooo diameters and magnification up to 50,000 diameters
have been achieved with this high precision microscope which
photographs or enables observation of both opaque. and
transparent preparations; in fact, polishing scratches
measuring, in width, but one-tenth the wavelength of light used
have been clearly distinguished. It is the opinion of both Dr.
Graton and Dr. Dane that some present-day lenses are really
capable of better resolution than claimed for them by their
manufacturers, it having been their experience to use objectives
exhibiting superior qualities of resolution over those of
identical medium and numerical aperture, proving that not only
have already available lenses surpassed their theoretical limits
of resolution, indicating that it might be possible to design
objectives with still greater numerical apertures, but that the
accepted theory regarding this resolution is sadly in need of
revision. Dr. Lucas 5 microscope utilizing an objective with a
numerical aperture of 1.60, for instance, in combination with
monohromnaphalene immersion fluid, also yields resolution up to
6,ooo diameters being, like the Graton-Dane scope, a high
precision instrument constructed with the idea of maintaining
absolute stability of parts. Dr. Lucas also has expressed doubt
as to the complete validity of the generally accepted theory of
resolution.
In
working with a high precision ultra-violet micro-camera, into
which a tri-color filter system has been incorporated, which he
has just recently perfected, Dr. Lucas is able obtain a minimum
magnification
Page 13
of
30,000 diameters and a maximum magnification of 60,000
diameters. \With this instrument it is possible to view living
cells and organisms, no staining or killing of organisms being
necessary, and Dr. Lucas has succeeded in obtaining excellent
photomicrographs (both still and motion pictures). Of special
significance to industry, for instance, is the ability of this
scope to demonstrate the size, shape, and reactions in motion
and affinity of the tiny particles of which rubber is compose
under varying conditions of temperature, etc., while its ability
to reveaI living rat and mouse sarcoma and carcinoma cells and
to demonstrate the development and behavior of the syphilitic
organism is of far more than average interest to medical
science.
England's
Dr. J. E. Barnard has succeeded in obtaining resolution up to
7,500 diameters with his ultra-dark-field scope in which he uses
a combined illuminator. In this, an outer system of glass acts
as the immersion dark-field illuminator while the inner
immersion system of quartz makes possible the passage of a
transmitted beam of light through the specimen. Both condensers
have the same focus, one for visible light, the other for
ultra-violet radiation, and both can be stopped out at will.
When1 for instance, bacteria are being observed, immersion
contact is made between the condenser and quartz slide, the
dark-field illuminator being used, thus revealing the bacteria
with visible light. When the dark-field illuminator is closed,
however, a beam of ultraviolet light may be directed up through
the quartz condenser and focused on the bacteria. The
object-glass, of course, has to be adjusted since it does not
possess the same focus for ultra-violet that it does for visible
light. Staining of specimens is thus unnecessary, making it
possible to secure photomicrographs of living minute organisms.
In
addition to these four microscopes, a fourth, belonging to the
Canadian Department of Mines and located at Ottawa, and almost
identical in principle and construction to that of Doctors Dane
and Graton, has demonstrated ability to attain equally high.
This,
like the scopes of Doctors Dane, Graton, and Lucas, is fitted
with a tube for visual observation although intended mainly for
microphotographical work in the field of metallurgy. It is Dr.
Graton's belief, however, that his instrument and that of Dr.
Dane might also be adaptable to the purposes of biological
research. Referring, in the description of their
"Precision, All Purpose Microcamera" (Journal of the
Optical Society of America), to the necessity or
"desirability" of "reexamining the classical
conception of the limit of useful magnification," Doctors
Dane and Graton have this to say:
"So
long as the makers accepted the conventional limit as valid and
had already attained it, there was little incentive toward
progress. But with that limit apparently surpassed, there is no
present knowledge as to how far ahead the true limit may lie. If
present-day objectives do substantially better than the 'limit'
for which they were designed, is it not reasonable to suppose
that effort to do better still may conceivably be
rewarded?"
To
such an inquiry there can be but one logical answer-an agreement
which, while perhaps not concurred in by all, must, for those
stimulated to more intense interest and effort by the
possibilities of uncovering new facts, pose further questions;
for, if the improvement of one part results in the improved
performance of the whole, is it not also reasonable to suppose
that additional changes of additional parts, yes, even changes
with respect to principle and method might likewise bear fruit?
THE
UNIVERSAL MICROSCOPE
It
is not only a reasonable supposition, but already, in one
instance, a very successful and highly commendable achievement
on the part of Dr. Royal Raymond Rife of San Diego, California,
who, for many
Page 14
years,
has built and worked with light microscopes which far surpass
the theoretical limitations of the ordinary variety of
instrument, all the Rife' scopes possessing superior ability to
attain high magnification with accompanying high resolution. The
largest and most powerful of these, the Universal '\~1icroscope,
developed in 1933, consists of 5,682 parts and is so-called
because of its adaptability in all fields of microscopical work,
being fully equipped with separate substage condenser units for
transmitted and monochromatic beam, dark-field, polarized, and
slit-ultra illumination, including also a special device for
crystallography. The entire optical system of lenses and prisms
as well as the illuminating units are made of block-crystal
quartz, quartz being especially trans-parent to ultra-violet
radiations.
The
illuminating unit used for examining the filterable forms of
disease organisms contains fourteen lenses and prisms, three of
which are in the high-intensity incandescent lamp, four in the
Risley prism, and seven in the achromatic condenser which,
incidentally, has a numerical aperture of 1.40. Between the
source of light and the specimen are subtended two circular,
wedge-shaped, block-crystal quartz prisms for the purpose of
polarizing the light passing through the specimen, polarization
being the practical application of the theory that light waves
vibrate in all planes perpendicular to the direction in which
they are propagated. Therefore, when light comes into contact
with a polarizing prism, it is divided or split into two beams,
one of which is refracted to such an extent that it is reflected
to the side of the prism without, of course, passing through the
prism while the second ray, bent considerably less, is thus
enabled to pass through the prism to illuminate the specimen.
When the quartz prisms on the Universal Microscope, which may be
rotated with vernier control through 360 degrees, are rotated in
opposite directions, they serve to bend the transmitted beams of
light at variable angles of incidence while, at the same time, a
spectrum is projected up into the axis of the microscope, or
rather a small portion of a spectrum since only a part of a band
of color is visible at any one time. However, it is possible to
proceed in this way from one end of the spectrum to the other,
going all the way from the infra-red to the ultra-violet. Now,
when that portion of the spectrum is reached in which both the
organism and the color band vibrate in exact accord, one with
the other, a definite characteristic spectrum is emitted by the
organism. In the case of the filter-passing form of the Bacillus
Typhosus, for instance, a blue spectrum is emitted and the plane
of polarization deviated plus 4.8 degrees. The predominating
chemical constituents of the organism are next ascertained after
which the quartz prisms are adjusted or set, by means of vernier
control, to minus 4.8 degrees (again in the case of the
filter-passing form of the Bacillus Typhosus) so that the
opposite angle of refraction may be obtained. A monochromatic
beam of light, corresponding exactly
Page 15
to the frequency
of the organism (for Dr. Rife has found that each disease
organism responds to and has a definite and distinct wavelength,
a fact confirmed by British medical research workers), is then
sent up through the specimen and the direct transmitted light,
thus enabling the observer to view the organism stained in its
true chemical color and revealing its own individual structure
in a field which is brilliant with light. The objectives used on
the Universal Microscope are a 1.12 dry lens, a 1.16 water
immersion, a 1.18 oil immersion, and a 1.25 oil immersion. The
rays of light refracted by the specimen enter the objective and
are then carried up the tube in parallel rays through twenty-one
light bends to the ocular, a tolerance of less than one
wavelength of visible light only being permitted in the core
beam, or chief ray, of illumination. Now, instead of the light
rays starting up the tube in a parallel fashion, tending to
converge as they rise higher and finally crossing each other,
arriving at the ocular separated by considerable distance as
would be the case with an ordinary microscope, in the Universal
tube the rays also start their rise parallel to each other but,
just as they are about to cross, a specially-designed quartz
prism is inserted which serves to pull them out parallel again,
another prism being inserted each time the rays are about ready
to cross. These prisms, inserted in the tube, which are adjusted
and held in alignment by micrometer screws of one hundred
threads to the inch in special tracks made of magnelium
(magnelium having the closest coefficient of expansion of any
metal to quartz), are separated by a distance of only thirty
millimeters. Thus, the greatest distance that the image in the
Universal is projected through any one media, either quartz or
air, is thirty millimeters instead of the i6o, 180, or 190
millimeters as in the empty or air-filled tube of an ordinary
microscope, the total distance which the light rays travel
zig-zag fashion through the Universal tube being 449
millimeters, although the physical length of the tube itself is
229 millimeters. It will be recalled, that if one pierces a
black strip of paper or cardboard with the point of a needle and
then brings the card up close to the eye so that the hole is in
the optic axis, a small brilliantly-lighted object will appear
larger and clearer, revealing more fine detail, than if it were
viewed from the same distance without the assistance of the
card. This is explained by the fact that the beam of light
passing through the card is very narrow, the rays entering the
eye, therefore, being practically parallel, whereas without the
card the beam of light is much wider and the diffusion circles
much larger. It is this principle of parallel rays in the
Universal Microscope and the resultant shortening of projection
distance between any two blocks or prisms plus the fact that
objectives can thus be substituted for oculars, these
"oculars" being three matched pairs of ten-millimeter,
seven-millimeter, and four-millimeter objectives in short
mounts, which make possible not only the unusually high
magnification
Page 16
and
resolution but which serve to eliminate distortion as well as
all chromatic and spherical aberration.
Quartz
slides with especially thin quartz cover glasses are used when a
tissue section or culture slant is examined, the tissue section
itself also being very thin. An additional observational tube
and ocular which yield a magnification of 1 ,800 diameters are
provided so that that portion of the specimen which it is
desired should be examined may be located and so that the
observer can adjust himself more readily when viewing a section
at a high magnification.
The
Universal stage is a double rotating stage graduated through 360
degrees in quarter minute arc divisions, the upper segment
carrying the mechanical stage having a movement of 40 degrees,
the body assembly which can be moved horizontally over the
condenser also having an angular tilt of 40 degrees plus or
minus. Heavily-constructed joints and screw adjustments maintain
rigidity of the microscope which weighs two hundred pounds and
stands twenty-four inches high, the bases of the scope being
nickel cast-steel plates, accurately surfaced, and equipped with
three leveling screws and. two spirit levels set at angles of 90
degrees. The coarse adjustment, a block thread screw with forty
threads to the inch, slides in a one and one-half dovetail which
gibs directly onto the pillar post. The weight of the quadruple
nosepiece and the objective system is taken care of by the
intermediate adjustment at the top of the body tube. The stage,
in conjunction with a hydraulic lift, acts as a lever in
operating the fine adjustment. A six-gauge screw having a
hundred threads to the inch is worked through a gland into a
hollow, glycerine-filled post, the glycerine being displaced and
replaced at will as the screw is turned clockwise or
anti-clockwise, allowing a five-to-one ratio on the lead screw.
This, accordingly, assures complete absence of drag and inertia.
The fine adjustment being seven hundred times more sensitive
than that of ordinary microscopes, the length of time required
to focus the Universal ranges up to one hour and a half which,
while on first consideration, may seem a disadvantage, is after
all but a slight inconvenience when compared with the many years
of research and the hundreds of thousands of dollars spent and
being spent in an effort to isolate and to look upon
disease-causing organisms in their true form.
Working
together back in '93' and using one of the smaller Rife
Microscopes having a magnification and resolution of 17,000
diameters, Dr. Rife and Dr. Arthur Isaac Kendall of the
Department of Bacteriology of Northwestern University Medical
School were able to observe and demonstrate the presence of the
filter-passing forms of Bacillus Typhosus. An agar slant culture
of the Rawlings strain of Bacillus Typhosus was first prepared
by Dr. Kendall and inoculated into six cubic centimeters of
"Kendall" K Medium, a medium rich in protein but poor
in peptone arid consisting of one hundred mg. of
Page 17
dried
hog intestine and 6 CC of tyrode .Solution (containing neither
glucose nor glycerine) which mixture is shaken well so as to
moisten the dried intestine powder and then sterilized in the
autoclave, fifteen pounds for fifteen minutes, alterations of
the medium being frequently necessary depending upon the
requirements for different organisms Now, after a period of
eighteen hours in this K Medium, the culture was passed through
a Berkefeld "N" filter, a drop of the filtrate being
added to another six cubic centimeters of K Medium and incubated
at 37 degrees centigrade. Forty-eight hours later this same
process was repeated, the "N" filter again being used,
after which it was noted that the culture no longer responded to
peptone medium, growing now only in the protein medium. Then
again, within twenty-four hours, the culture was passed through
a filter-the finest Berkefeld "W" filter, a drop of
the filtrate was once more added to six cubic centimeters of K
Medium and incubated at 37 degrees centigrade, a period of three
(lays elapsing before the culture was transferred to K Medium
and yet another three days before a new culture was prepared.
Then, viewed under an ordinary microscope, these cultures were
observed to be turbid and to reveal no bacilli whatsoever. When
viewed by means of dark-field illumination and oil immersion
lens, however, the presence of small, actively-motile granules
was established, although nothing at all of their individual
structure could be ascertained. Another period of four days was
allowed to elapse before these cultures were transferred to K
Medium and incubated at 37 degrees centigrade for twenty-four
hours when they were then examined under the Rife Microscope
where, as was mentioned earlier, the filterable typhoid bacilli,
emitting a blue spectrum, caused the plane of polarization to be
deviated plus 4.8 degrees. Then when the opposite angle of
refraction was obtained by means of adjusting the polarizing
prisms to minus 4.8 degrees and the cultures illuminated by a
monochromatic beam coordinated in frequency with the chemical
constituents of the typhoid bacillus, small, oval,
actively-motile, bright turquoise-blue bodies were observed at a
magnification of 5,000 diameters, in high contrast to the
colorless and motionless debris of the medium. These
observations were repeated eight times, the complete absence of
these bodies in uninoculated control K Media also being noted.
To
further confirm their findings, Doctors Rife and Kendall next
examined eighteen-hour old cultures which had been inoculated
into K Medium and incubated at 37 degrees centigrade, since it
is just at this stage of growth in this medium and at this
temperature that the cultures become filterable. And, just as
had been anticipated, ordinary (dark-field examination revealed
unchanged, long, actively-motile bacilli having granules within
their substance; and free-swimming, actively-motile granules;
while under the Rife Microscope were demonstrated the same long,
unchanged almost colorless bacilli; bacilli, prac-
Page 18
tically
colorless, inside and at one end of which was a turquoise-blue
granule resembling the filterable forms of the typhoid baciIlus;
and free-swimming, small, oval; actively-motile, turquoise-blue
granules. By transplanting the cultures of the filter-passing
organisms or virus into a broth, they were seen to change over
again into their original rod-like forms.
At
the same time these findings of Doctors Rife and Kendall were
confirmed by Dr. Edward C. Rosenow of the Mayo Foundation, the
magnification with accompanying resolution of 8,ooo diameters of
the Rife Microscope, operated by Dr. Rife, was checked against a
dark-field oil immersion scope operated by Dr. Kendall and an
ordinary 2 mm. oil immersion objective, X 10 ocular, Zeiss scope
operated by Dr. Rosenow at a magnification of 900 diameters.
Examinations of gram and safranin stained films of cultures of
Bacillus Typhosus, gram and safranin stained films of cultures
of the streptococcus from poliomyelitis, and stained films of
blood and of the sediment of the spinal fluid from a case of
acute poliomyelitis were made with the result that bacilli,
streptococci, erythrocytes, polymorphonuclear leukocytes, and
lymphocytes measuring nine times the diameter of the same
specimens observed under the Zeiss scope at a magnification and
resolution of 900 diameters, were revealed with unusual clarity.
Seen under the dark-field microscope were moving bodies presumed
to be the filterable turquoise-blue bodies of the typhoid
bacillus which, as Dr. Rosenow has declared in his report
("Observations on Filter-Passing Forms of Eberthella
Typhi-Bacillus Typhosus-and of the Streptococcus from
Poliomyelitis," Proceedings of the Staff Meetings of the
Mayo Clinic, July 13, 1932), were so "unmistakably
demonstrated" with the Rife Microscope, while under the
Zeiss scope stained and hanging drop preparations of clouded
filtrate cultures were found to be uniformly negative. With the
Rife Microscope also were demonstrated brownish-gray cocci and
diplococci in hanging drop preparations of the filtrates of
streptococcus from poliomyelitis. These cocci and diplococci,
similar in size and shape to those seen in the cultures although
of more uniform intensity, and characteristic of the medium in
which they had been cultivated, were surrounded by a clear halo
about twice the width of that at the margins of the debris and
of the Bacillus Typhosus. Stained films of filtrates and
filtrate sediments examined under the Zeiss microscope, and
hanging drop, dark-field preparations revealed no organisms,
however. Brownish-gray cocci and diplococci of the exact same
size and density as those observed ~ the filtrates of the
streptococcus cultures were also revealed in hanging drop
preparations of the virus of poliomyelitis under the Rife
Microscope, while no organisms at all could be seen in either
the stained films of filtrates and filtrate sediments examined
with the Zeiss scope nor in hanging drop preparations examined
by means of the dark-field. Again using the Rife Microscope
Page 19
At
a magnification of 8,ooo diameters, numerous nonmotile cocci and
diplococci of a bright-to-pale pink in color were seen in
hanging drop preparations of filtrates of Herpes encephalitic
virus. Although these were observed to be comparatively smaller
than the cocci and diplococci of the streptococcus and
poliomyelitic viruses, they were shown to be of fairly even
density, size, and form and surrounded by a halo. Again, both
the dark-field and Zeiss scopes failed to reveal any organisms,
and none of the three microscopes disclosed the presence of such
diplococci in hanging drop preparations of the filtrate of a
normal rabbit brain. Dr. Rosenow has since revealed these
organisms with the ordinary microscope at a magnification of
1,000 diameters by means of his special staining method and with
the Electron Microscope at a magnification of 12,000 diameters.
Dr. Rosenow has expressed the opinion that the
Page 20
inability
to see these and other similarly revealed organisms is due, not
necessarily to the minuteness of the organisms, but rather to
the fact that they are of non-staining hyaline structure.
Results with the Rife Microscopes, he thinks, are due to the
"ingenious methods employed rather than to excessively high
magnification." He has declared also, in the report
mentioned previously, that examination under the Rife microscope
of specimens containing objects visible with the ordinary
microscope, leaves no doubt of the accurate visualization of
objects or particulate matter by direct observation it the
extremely high magnification obtained with this instrument.
Exceedingly
high powers of magnification with accompanying high powers of
resolution may be realized with all of the Rife Microscopes one
of which, having magnification and resolution up to 8,000
diameters, is now being used at the British School of Tropical
Medicine in England. In a recent demonstration of another of the
smaller Rife scopes (May 6th, 1942) before group of doctors
including Dr. J. H. Renner of Santa Barbara, California; Dr.
Roger A. Schmidt of San Francisco, California; Dr. Lois Bronson
Slade of Alameda, California; Dr. Lucile B. Larkin of
Bellingham, Washington; Dr. E. F. Larkin of Bellingham,
Washington; and Dr. W. J. Gier of San Diego, California, a Zeiss
ruled grading was examined, first under an ordinary commercial
microscope equipped with a 1.8 high dry lens and X 10 ocular and
then under the Rife Microscope. Whereas fifty lines were
revealed with the commercial instrument and considerable
aberration, both chromatic and spherical noted, only five lines
were seen with the Rife scope these five lines being so highly
magnified that they occupied the entire field, without any
aberration whatsoever being apparent. Dr. Renner, in a
discussion of his observations, stated that the entire field to
its very edges and across the center had a uniform clearness
that was not true in the conventional instrument. Following the
examination of the grading, an ordinary unstained blood film was
observed under the same two microscopes. In this instance, one
hundred cells were seen to spread throughout the field of the
commercial instrument while but ten cells filled the field of
the Rife scope.
The
Universal Microscope, of course, is the most powerful Rife
scope, possessing a resolution of 31,000 diameters and
magnification of 60,000 diameters. With this it is possible to
view the interior of the "pin point" cells, those
cells situated between the normal tissue cells and just visible
under the ordinary microscope, and to observe the smaller cells
which compose the interior of these pin point cells. When one of
these smaller cells is magnified, still smaller cells are seen
within its structure. And when one of the still smaller cells,
in its turn, is magnified, it, too, is seen to be composed of
smaller cells. Each of the sixteen times this process of
magnification and resolution can be repeated, it is demonstrated
that there are smaller cells within the
Page 21
smaller
cells, a fact which amply testifies as to the magnification and
resolving power obtainable with the Universal Microscope.
More
than 20,000 laboratory cultures of carcinoma, were grown and
studied over a period of seven years by Dr. Rife and his
assistants in what, at the time, appeared to be' a fruitless
effort to isolate the filter-passing form, or virus, which Dr.
Rife believed to be present in this condition. Then, in 1932,
the reactions in growth of bacterial cultures to light from the
rare gasses was observed, indicating a new approach to the
problem. Accordingly, blocks of tissue one-half centimeter
square, taken from an unulcerated breast carcinoma, were placed
in triple-sterilized K Medium and these cultures incubated at 37
degrees centigrade. When no results were forthcoming, the
culture tubes were placed in a circular glass loop filled with
argon gas to a pressure of fourteen millimeters, and a current
of 5,OOQ volts applied for twenty-four hours, after which the
tubes were placed in a two-inch water vacuum and incubated at 37
degrees centigrade for twenty-four hours. Using a specially
designed 1.12 dry lens, equal in amplitude of magnification to
the 2 mm. apochromatic oil immersion lens, the cultures were
then examined tinder the Universal Microscope, at a
magnification of 10,000 diameters, where very-much animated,
purplish-red, filterable forms, measuring less than
one-twentieth of a micron in dimension, were observed. Carried
through fourteen transplants from K Medium to K Medium, this B.
X. virus remained constant, inoculated into four hundred and
twenty-six Albino rats, tumors "with all the true pathology
of neoplastic tissue" were developed. Experiments conducted
in the Rife Laboratories have established the fact that these
characteristic diplococci are found in the blood monocytes in 92
per cent. of all cases of neoplastic diseases. It has also been
demonstrated that the virus of cancer, like the viruses of other
diseases, can be easily changed from one form to another by
means of altering the media upon which it is grown. With the
first change in media, the B. X. virus becomes considerably
enlarged although its purplish-red color remains unchanged.
Observation of the organism with an ordinary microscope is made
possible by a second alteration of the media. A third change is
undergone upon asparagus base media where the B. X. virus is
transformed from its filterable state into cryptomyces
pleomorphia fungi, these fungi being identical morphologically
both macroscopically and microscopically to that of the orchid
and of the mushroom. And yet a fourth change may be said to take
place when this cryptomyces pleomorphia, permitted to stand as a
stock culture for the period of metastasis, becomes the
well-known mahogany-colored Bacillus Coli.
It
is Dr. Rife's belief that all microorganisms fall into one of
not more than ten individual groups (Dr. Rosenow has stated that
some of the viruses belong to the group of the streptococcus)
and that any alteration of artificial media or slight metabolic
variation in tissues will
Page 22
induce
an organism of one group to change over into any other organism
included in that same group, it being possible, incidentally, to
carry such changes in media or tissues to the point where the
organisms fail to respond to standard laboratory methods of
diagnosis. These changes can be made to take place in as short a
period of time fourty-eight hours.
For instance, by altering the media-four parts per million per
volume-the pure culture of mahogany-colored bacillus Coli
becomes the turquoise-blue Bacillus Typhosus. Viruses or
primordial cells of organisms which would ordinarily require an
eight-week incubation period to attain their filterable state,
have been shown to produce disease within three days' time,
proving Dr. Rife's contention that the incubation period of a
microorganism is really only a cycle of reversion. He states:
"In reality, it is not the bacteria themselves that produce
the disease, but we believe it is the chemical constituents of
these microorganisms enacting upon the unbalanced cell
metabolism of the human body that in actuality produce the
disease. We also believe if the metabolism of the human body is
perfectly balanced or poised, it is susceptible to no
disease."
In other
words, the human body itself is chemical in nature, being
comprised of many chemical elements which provide the media upon
which the wealth of bacteria normally present in the human
system feed. These bacteria are able to reproduce. They, too,
are composed of chemicals. Therefore, if the media upon which
they feed, in this instance the chemicals or some portion of the
chemicals of the human body, becomes changed from the normal, it
stands to reason that these same bacteria, or at least certain
numbers of them, will also undergo a change chemically since
they are now feeding upon a media which is not normal to them,
perhaps being supplied with too much or too little of what they
need to maintain a normal existence. They change, passing
usually through several stages of growth, emerging finally as
some entirely new entity as different morphologically as are the
caterpillar and the butterfly (to use an illustration given us).
The majority of the viruses have been definitely revealed as
living organisms, foreign organisms it is true, but which once
were normal inhabitants of the human body-living entities of a
chemical nature or composition.
Under the
Universal Microscope disease organisms such as those of
tuberculosis, cancer, sarcoma, streptococcus, typhoid,
staphylococcus, leprosy, hoof and mouth disease, and others may
be observed to succumb when exposed to certain lethal
frequencies, coordinated with the particular frequencies
peculiar to each individual organism, and directed upon them by
rays covering a wide range of waves. By means of a camera
attachment and a motion picture camera not built into the
instrument, many "still" micrographs as well as
hundreds of feet of motion picture film bear witness to the
complete life cycles of numerous organisms. It should be
emphasized, perhaps, that invariably the same organisms refract
the same colors when stained by means of the monochromatic beam
of illumination on the Universal Microscope, regardless of the
media upon which they are grown. The virus of the Bacillus
Typhosus is always a turquoise-blue, the Bacillus Coli always
mahogany-colored, the Mycobacterium li prae always a ruby shade,
the filter-passing form or virus of tuberculosis always an
emerald green, the virus of cancer always a purplish-red, and so
on. Thus, with the aid of this microscope, it is possible to
reveal the typhoid organism, for instance, in the blood of a
suspected typhoid patient four and five days before a Widal is
positive. When it is desired to observe the flagella of the
typhoid organism, Hg salts are used as the media to see at a
magnification of 10,000 diameters.
Page 23
In
the light of the amazing results obtainable with this Universal
Microscope and its smaller brother-scopes, there can be no doubt
of the ability of these instruments to actually reveal any and
all microorganisms according to their individual structure and
chemical constituents.
With the
aid of its new eyes-the new microscopes, all of which are
continually being improved-Science has at last penetrated beyond
the boundary of accepted theory and into the world of the
viruses with the result that we can look forward to discovering
new treatments and methods of combating the deadly organisms for
Science does not rest.
To
Dr. Karl K. Darrow, Dr. John A. Kolmer, Dr. William P. Lang, Dr.
L. Marton, Dr. 3. H. Renner, Dr. Royal R. Rife, Dr. Edward C.
Rosenow, Dr. Arthur W. Yale, and Dr. V. K. Zworykin, we wish to
express our appreciation for the help and information so kindly
given us and to express our gratitude, also, for the interest
shown in this effort of bringing to the attention of more of the
medical profession the possibilities offered by the new
microscopes.
REFERENCES.
ANDERSON
T. F., AND STANLEY, W. M.: "A Study by Means of the
Electron Microscope of the Reaction Between Tobacco
Mosaic Virus and its Antiserum," Jour. Biol. Chem., 139:
No. 1, 339-344, 1941.
ANDRADE,
E. N. DA C.: "The Mechanism of Nature," G. Bell &
Sons, Ltd., London, 1932.
APPERLY,
F. L.: "Relation of Solar Radiation in North America,"
Cancer Research, I: 191-195, March '94'.
BATEMAN,
3. B., CALKINS, H. E., AND CHAMBERS, L. A.: "Optical Study
of the Reaction Between Transferred Monolayers of Lancefield's
'M' Substance and Various Antisera,"
Jour.
Immun., 41: 321-341, 1941.
BURTON,
E. F.: "The Electron Microscope," Proc. Roy.
Can. Insi., 5: Series 3A, Feb.10, 1940.
CORRINGTON,
JULYAN D.: "Under the Microscope," Nat. Mag., 34: 117-120,
Feb.1941.
DARROW,
KARL, K.: "The Renaissance of Physics," Macmillan Co.,
1939.
DAVIS,
WATSON: "Exploring A New World," Science News Letter,
38: 227-230, Oct.12, 1940.
Same:
Abr. with title, "30,000 Times Life-Size," Readers
Digest, 37: 13-16, Nov.1940.
DUNN,
H. H.: "Movie New Eye of Microscope in War on Germs,"
Pop. Science, 118: 27, June 1931.
ELDRIDGE,
JOHN A.: "The Physical Basis of Things," 1934.
"Electron
Microscope Magnifies Invisible World 25,000 X." Life, 8:
54, April 29,1940.
"Electron
Microscope Shows 'Strep' Has Outer Membrane," Science News
Letter, 38: 301, Nov. 9,1940.
"Filterable
Germ Forms Seen with New Super-Microscope," Science News
Letter, Dec. 12, 1931.
"Filterable
Germs Need not be Extremely Small," News Science Letter,
Nov.12, 1932.
FRIEDMAN,
JOSEPH S.: "New Electron Microscope," Amer. Photography,
35: 368-369, Mar. '94'.
GAGE,
SIMON HENRY: "The Microscope," 16th. and 17th. Ed.,
Comstock Pub. Co., Inc., 1936-1941.
GRATON,
L. C., AND DANE, E. C.: "Precision, All Purpose
Microcamera," Jour. Opt. Soc. Amer., 27: 355-376,
Nov.1937.
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