Note: Descriptions are shown in the official language in which they were submitted.
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INVERTED DARKFIELD CONTRAST MICROSCOPE AND METHOD
FIELD OF THE INVENTION
The present invention relates to microscopes and methods of obtaining images
therewith. More particularly, the present invention relates to a method of
obtaining images with
Inverted Darkfield Contrast (IDC) microscopes and a novel IDC microscope.
BACKGROUND OF THE INVENTION
For many years light microscopes have been considered a mature technology.
While
there have been notable attempts to extend the capabilities of the light
microscope, to date such
attempts have not achieved substantial gains in performance and have generally
been obtained at
significantly increased costs. Vibrations in microscopes have been known as a
major factor
contributing to the limit on resolving power. Vibrations in the microscope
frame have previously
been addressed by building super rigid or heavy frames, or by constructing
horizontal microscopes
on massive optical bench-style frames. Other attempts to improve the vibration
performance have
used passive or active vibration damping tables, feet or platforms.
The generation of image contrast in microscopes is an area where there has
been
considerable work carried out in the past. Attempts to increase the contrast
of observed biological
samples have resulted in many new methods such as phase contrast, interference
contrast, Hoffman
modulation contrast, differential interference contrast, polarized light
microscopy, darkfield
microscopy and fluorescent microscopy. The challenge of generating image
contrast at the extreme
limit of resolution yielded such techniques as high power immersion darkfield,
and
ultramicroscopic illumination. Phase and interference contrast techniques
introduced artifacts,
some of which were asymmetrical, which made the images difficult to relate to
the real structure of
the samples being viewed. Darkfield and fluorescent techniques presented image
information in a
form that is most unfamiliar to visual capabilities, much in the same way that
we are unable to
extract information from a photographic or electronic "negative" image.
Attempts to gain more information about cells in real time has yielded
confocal
microscopy which uses high power laser light sources which scan the sample
area to build a final
image of the sample, and newer masked confocal techniques that can build
higher speed images of
live samples. In general, the frame / field rate of the confocal systems is
too slow for studying the
high speed motion of many components in biological systems since they exhibit
high speed motion.
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Attempts to yield high resolution have been based on the formula for
microscopic
resolution first developed by Ernst Abbe, resolution limit = wavelength of
light/ (k x numerical
aperture of the objective). Values for k ranging from 1.6 to 2 have been
accepted for over 50 years
but the inventor's recent work suggests the value of k can be lowered and
needs to be more fully
studied when applied to improved optical systems with new methods of
illumination and imaging
means.
As microscope systems have become more complex, more glass surfaces created
more light loss due to transmission losses in the glass elements, internal
reflection and stray light.
The stray light contributed to poor contrast and the internal reflections and
transmission losses,
together with the stray light, meant that progressively more powerful light
sources were needed to
produce useable image brightness. These high powered sources must propagate
the light at high
fluxes through the sample space since most of the lossy components are between
the sample and the
imaging means. Modern binocular and trinocular systems with their attendant
prisms, mirrors and
lenses are particularly inefficient and require higher light levels.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel IDC microscope and
a
novel method of obtaining images with an IDC.
According to a first aspect of the present invention, there is provided a
novel method
of achieving contrast for microscopical imaging of preparations of living
cells and other types of
objects is described along with improvements to microscopes. This method
combines the
traditional darkfield illumination technique with electronic image inversion
(converting a positive
to a negative image) and other improvements to further enhance the contrast
and resolution of the
final image. The method is referred to herein as Inverted Darkfield Contrast
and is believed to be
particularly suitable for viewing live cells in real time with no staining or
preparation.
The embodiments shown herein are primarily based on a video microscope in
which
image resolution, contrast and optical efficiency are optimized. In
microscopes in accordance with
the present invention there is usually no intervening binocular or trinocular
arrangement or eyepiece
between the objective and the imaging system, which can be any type of imaging
means including
film cameras, analog or digital video cameras or image intensifiers. The
microscope system can
use a pre-focused and aligned lamp and reflector to direct a larger than usual
portion of the light
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from the lamp into the illuminating beam. The illuminating beam is directed
through a beam
expander which controls the diameter of the illuminating beam while
maintaining parallel rays of
light. The illuminating beam passes through apertures to control stray light.
Careful attention is paid to controlling the illuminating wavelengths of light
to
improve the resolution of the microscope. In particular, all the non-visible
wavelengths in the
ultraviolet (UV) and infrared (IR) portion of the spectrum are preferably
eliminated to improve
image quality. The rays of light leaving the objective are also passed through
apertures and baffle
tube(s) to reduce stray light and enhance contrast. Anti-vibration means are
also provided to
control the motion of the objective, relative to the sample being viewed, and
the position of the
imaging device relative to the objective. Control of stray light in the
objective and in the coupler
between the objective and the imaging device also help to improve contrast and
resolution.
The signal from the imaging device is inverted to form the negative of the
normal
image. In this way the traditional darkfield image appears as a high contrast
brightfield image in
the final monitor or computer display.
The present invention comprises a variety of mechanical and optical
improvements
to a microscope in order to achieve Inverted Darkfield Contrast (IDC). More
specifically, a video
microscope is provided which can include improvements to the illumination
system, the condenser,
the slide holder, the objectives, the tube, the microscope stand and the image
acquiring system to
produce a novel IDC microscope.
The present invention provides a method for obtaining high contrast images of
living
biological samples such as cells in real time with no staining or
fluorochemistry required. The
method is applicable to imaging a variety of materials, substances and
structures, including cells,
internal cellular structures, bacteria, viruses, fungi and plant materials.
The present invention also
includes improvements in microscope technology including improvements to stand
design,
illuminators, condensers, objectives, imaging systems and to video processing.
While the concept of darkfield imaging is not new and the use of video
positive to
negative inversion is known in the television broadcast special effects field,
the present invention is
the first application of these unrelated techniques to obtain high contrast
images of samples such as
living biological material. The present invention provides particular
advantages as it can provide
images which look like stained biological materials, so that biologists can
readily interpret and
accept the information that the images present, without requiring the staining
of the imaged
samples. The present invention can improve the contrast, resolution and speed
of acquisition of the
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image, without significantly increasing the cost or complexity of the
microscope.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the present invention will now be described, by way
of
example only, with reference to the attached Figures, wherein:
Figure 1 shows an embodiment of an IDC microscope in accordance with the
present
invention;
Figure 2 shows an embodiment of the arrangement of the video system of the IDC
microscope;
Figure 3 shows an embodiment of a piezoelectrically actuated brace; and
Figure 4 shows a cross section of a typical objective used in an IDC
microscope in
accordance with the present invention; and
Figure 4a shows the detail of the area identified at A in Figure 4.
DETAILED DESCRIPTION OF THE INVENTION
The present invention forms a darkfield image with a high numerical aperture
(NA)
optical system and electronically inverts the digitized darkfield image to
produce a negative image
of the darkfield image. The negative image is an apparent brightfield image,
with very high
contrast and resolution.
While it is possible to implement the method of the present invention using
standard
microscope illuminators, it is presently preferred that the light source
employed with the present
invention be considered on the basis of a "photon budget", where the intended
destination of each
photon from the light source is mapped and accounted for in the design of the
IDC microscope. In
order to achieve this goal, in a presently preferred embodiment of the
invention, the light source is
selected and constructed as follows.
In conventional microscopes, tungsten, tungsten halogen, quartz halogen, or
arc light
sources are employed. These light sources are not well controlled in terms of
the position of the
light emitting surface of the source and consequently there is usually a means
for centering the light
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emitting surface in the X, Y and Z directions with respect to the optical path
of the microscope.
In contrast, in the presently preferred embodiment of the invention
illustrated in
Figure 1, the microscope indicated generally at 10, can employ a light source
14 in which the exact
location of the light emitting component is exactly controlled by the body of
light source 14 and/or
5 the socket it is mounted in. This eliminates the need for a centering
mechanism for light source 14
and ensures that substantially the highest possible intensity and geometrical
control of the beam and
repeatability is achieved. Suitable examples of such light sources are the
Welch Allen lamps for
medical applications, the ILC arc lamps, the GE and Sylvania prefocused lamps
and other, similar,
light sources. To the best of the present inventor's knowledge, to date these
light sources have not
been employed with light microscopes.
In microscope 10, light source 14, which is supplied with the necessary power
from
a suitable power supply 16, is mounted such that as much light as possible
from the light emitting
surface, or surfaces, is focused by a suitable illuminator focus means, such
as a mirror 18 behind
light source 14 and/or a lens 22 in front of light source 14. The light from
the back of light source
14 is focused back onto, or adjacent to, the emitting surface(s) of light
source 14 by mirror 18. The
light from the front of light source 14 and that returned by mirror 18 is
focused into a collimated
beam by lens 22, or a set of lenses, in front of light source 22. Suitable
apertures 26, baffles 30 or
tubular structures (not shown) are employed to ensure that the light from lens
22 is substantially
completely collimated. It is desired to collimate the light from lens 22 so
that little or no off-axis
light enters the condenser system, described below, of microscope 10. Such off-
axis light would
become "stray light" in the imaging optics and would degrade the contrast of
the final image.
As most light sources emit light which is outside the range of human vision,
and the
corrected range of microscope optics, a filter means 34 is provided in the
path of the illuminating
beam to filter the light to correspond as closely as possible to the range of
wavelengths for which
the optics of microscope 10 are designed. Filter means 34 can be included
anywhere in the
illumination beam path between the light source 14 and the final optics of the
condenser 38 and
filter means 34 can consist of the one or more heat filters such as Schott KG1
or KG5 glass, and can
include additional interference filters to attenuate the red or blue end of
the light spectrum, and can
exclude the ultraviolet portion of the spectrum with Schott WG or GG series
filters, for example.
By eliminating the infrared portion of the spectrum, heating of the sample
with its attendent impact
on living samples is greatly reduced. Eliminating the high energy ultraviolet
light from the light
reaching the sample means that samples are not subjected to as much DNA,
cellular and bleaching
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damage. This means that samples can be maintained on the microscope for longer
periods of time
during continuous study. It also means that the samples being studied are not
subject to the
abnormal levels of infrared and ultraviolet from the usual sources used in
microscopes which are
generally incandescent, metal halogen, or xenon or mercury arc sources, as
compared to the ratio of
infrared to visible to ultraviolet in the light coming from the sun, It is an
important feature of this
microscope that living samples are exposed only to ratios of infrared to
visible and ultraviolet to
visible at or less than ratios that samples would have been exposed to in
nature. This feature means
that samples behave in a fashion which is more closely analogous to their
behavior in their natural
environment.
By restricting the wavelengths of light present in the illuminating beam it is
possible
to operate the objective 40 of microscope 10 with light which forms a higher
resolution image of
the object due to the matching of the light to the design specifications of
objective 40 of microscope
10. In this way, the light passing through the sample can be limited to match
the best spherical and
chromatic aberration correction points of the objective. Typically the light
would be limited to two
wavelength regions for an achromatic objective or three wavelength regions for
an apochromatic
objective. This limitation on the wavelength of the light further reduces
sample heating and non
thermal effects.
It is contemplated that non plan optics will generally give the best images
when used
in this invention. This is because there are no trade offs for flatness of
field and the IDC system
uses only a small portion of the inside area of the total image. Accordingly
flatness of field is not
such a concern as it would be in other microscope systems which utilize the
full field of view of the
objective.
Where it is desirable to include fluorescence capability for microscope 10, a
position
can be provided in filter means 34 for an illuminating filter which limits the
illuminating beam
wavelengths to only those wavelengths that are important for exciting the
fluorophores being used
with the sample. In this case, the substrate of this filter should be kept as
thin a possible so that the
ray path of the illuminating beam is disrupted as little as possible. As the
method of illumination in
the present invention is darkfield, fluorescent imaging can be applied to this
method with almost the
same quality of results as with reflected light microscopy, even though the
image from the IDC
microscope appears to be and is nominally a "brightfield" transmitted light
technique. In order to
match the illuminating beam to characteristics of condenser 38 being employed,
additional optical
systems are included. Specifically, the collimated illumination beam from
light source 14 passes
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through one or both of two types of optical systems. The first type of optical
system is operable to
modify the illuminating beam dimension to match the optical requirements of
condenser 38. This
system can be a system of fixed lenses 42 and 46 or a zoom lens device (not
shown), either of
which operate to supply substantially the highest possible amount of light
from illumination source
14 to condenser 38 in a beam geometry selected to take full advantage of the
characteristics of
condenser 38.
When a darkfield condenser is employed, a parallel beam of light can be the
most
advantageous while in a conventional brightfield condenser, a converging beam
of light is desired
where the converging beam presents the image of the filament of the lamp at
the back focal plane of
the condenser to achieve Kohler illumination. If desired, the illumination
beam can pass through a
second optical system (not shown) to reshape the illuminating beam to achieve
Kohler illumination,
as is well known in the art.
Condenser 38 can be a high numerical aperture darkfield condenser of any type,
as is
known to those of skill in the art. The design of condenser 38 should create
an inner and outer cone
of illumination with a numerical aperture to match or exceed the optical
characteristics of the
objective 40 being employed. The presently preferred numerical apertures for
condenser 38 are
1.27 for the inner cone and 1.33 for the outer cone for most biological
applications, although for
lower powered objectives with lower numerical apertures a darkfield condenser
of lower NA can be
used. This is illustrated by employing a x63 objective with an NA of 0.7 and a
condenser of inner
cone NA of 0.71 and outer cone of NA 0.75.
As mentioned above, for many biological applications, a numerical aperture of
1.27
is presently preferred for the inner cone so that objectives of 1.25 numerical
aperture can be used
without requiring additional stops or irises to control their numerical
aperture and the contrast of the
darkfield effect. A numerical aperture of 1.33 is similarly preferred for the
outer cone to match the
index of refraction of aqueous media. As will be apparent to those of skill in
the art, for higher
index media or for highlighting high index materials that are directly in
contact with the slide, then
it can be preferred to employ in condenser 38 an aperture of 1.4 or greater
for the outer cone. For
"extreme" applications, and where the characteristics of the media surrounding
the sample and the
sample itself allow, it is presently preferred to employ a condenser 38 with a
numerical aperture of
1.42 for the inner cone and of 1.47 or higher for the outer cone. This allows
only those objects with
an index of refraction greater than 1.4 and which are in intimate contact with
the microscope slide,
to be highlighted against a very black background since the only light which
can pass into the
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sample when it is mounted in an aqueous media, is the light which flows into
the sample at the area
of contact with the slide. The sample objects thus appear to be luminous
against a completely dark
background. This mode allows the use of 1.4 NA objectives for the highest
possible resolution.
The drawbacks to this method are that any objects, either floating in the
mounting media or not
optically connected to the portion of the sample which is optically connected
to the slide will
disappear giving a false picture of the complete environment of the sample and
possibly loosing
some of the fine detail of the sample itself; and that objects can seemingly
completely disappear
without a trace if they suddenly loose contact with the slide.
One of the reasons to reduce the numerical aperture of the outer illuminating
cone in
aqueous applications is to limit the stray light which otherwise results when
a portion of the
illuminating cone from condenser 38 is reflected by total internal reflection
at the glass-water
interface of the sample back into condenser 38 where it becomes stray light.
Alternatively,
returning stray light can be trapped and absorbed in light traps or dumps
created by suitably baffled
or designed surface geometries.
The presently preferred types of designs for condenser 38 include the Zeiss
ultra-
dark field condenser, the older design Leitz darkfield condenser for the oil
immersion use, or
current production LOMO high numerical aperture darkfield condensers with an
inner NA of at
least 1.2.
It is presently preferred that condenser 38 employ the cone darkfield
illuminator, or
the coaxial darkfield/brightfield illuminator, both of which were designed
after the work of the J. E.
Barnard, circa 1933 and 1925 respectively and which are described in various
papers and
publications. In particular, in the cone condenser illustrated in Figure 1,
the illumination beam
passes through a conical prism 50 which forms an angled, but still collimated,
annulus of light.
This annulus is reflected off the surface of a circular mirror ring 54 which
focuses the light to a
hollow cone of the desired geometry. The elements of condenser 38 are
contained in a suitable
housing 58.
The illumination beam leaving condenser 38 passes through a spherical lens 62
in
such a way that the rays from the surface of mirror ring 54 pass through the
surface of lens 62 at
right angles and are undeviated. As condenser 3 8 is achromatic, it can be
employed equally well
for infrared, visible or ultraviolet light imaging applications.
The illumination beam from condenser 38 passes through the stage 66 of
microscope
10 and the slide 70 which supports the sample/object 74 being imaged. In most
circumstances,
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sample 74 will be covered by a cover slip 78. Due to the high numerical
apertures employed,
condenser 38 is preferably connected to slide 70 by a film of immersion oil as
is well known to
those of skill in the art.
Microscopes have historically been constructed with C-shaped frames with the
objective and eye-piece at the upper end of the frame and light source and
stage at the lower end of
the frame. The present inventor has determined that, while convenient to use
and manufacture,
conventional C-shaped frames suffer from disadvantages in that these frames
are susceptible to
undesired vibrations, and in fact are shaped and surprisingly act much like
tuning forks. It has been
found that external vibrations from any source and of virtually any frequency
tend to excite the
tuning fork shape of the conventional C-shaped frame to vibrate at its own
resonant frequency and
related harmonics, and this can distort the image resolved by the microscope.
These disadvantages
are particularly exacerbated with the present invention which otherwise can
allow microscope 10 to
resolve objects smaller than 250 nanometers, or less, and to detect objects as
small as less than 50
nanometers. Accordingly, it is preferred to attenuate vibration of the
microscope frame such that
undesired movement of objective 40 relative to the sample 74 being imaged is
inhibited.
The present inventor has determined two approaches to attenuating or
eliminating
this undesired vibration. The presently preferred first approach is to include
or add braces 78 which
connect the head of the microscope 82 to the base 86 of microscope 10. Braces
78 are attached to
microscope 10 along the vertical optical axis and on either side of stage 66
of microscope 10.
Braces 78 can be fabricated, machined or cast and are preferably made of a
material or materials,
such as aircraft aluminum alloys, or steel alloys, which have a relatively low
elasticity and tendency
to vibrate. In some cases it may be desired to construct the braces as
composites or sandwiched
layers of different materials in order to further stiffen the brace and reduce
the tendency to vibrate.
Preferably, braces 78 are designed to have as little resonant vibration as
possible and, of the
vibration which can not be eliminated, braces 78 are designed such that their
resonant frequency is
not a harmonic or sub-harmonic of the fundamental frequency of the vibration
of the microscope C-
shaped frame. In this manner the vibration of each of the frame and braces 78
tend to damp the
vibrations of the other.
The method of designing the braces is to first characterize the modes of
vibration of
the C-frame as completely as possible, with the full range of accessories that
may be used with the
microscope (since the vibration can vary with the accessories used). Once the
vibration modes are
understood, then the braces are designed to reduce the vibration, and to
attempt to make whatever
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vibration remains "common-mode so that all components of the microscope
vibrate in phase so
there is little or no "net" vibration from the point of the imaging means
relative to the object in
typical use.
Another approach to eliminating the vibration in a light microscope is to
employ a
5 tubular design for the frame of microscope 10, wherein the tube surrounds
stage 66 of microscope
10 in much the same way as the design of conventional scanning and
transmission electron
microscope sample chambers and columns. Such a tubular design can virtually
eliminate the Z axis
vibrations of objective 40 relative to the sample 74. While the tubular design
severely limits access
to the sample area, the gain in vibration performance is considerable and can
be well worth the
10 inconvenience in cases where the best possible resolution is desired.
Objective 40 of microscope 10 can be designed as a fixed focal length
objective to
produce a completely corrected image at the first image plane of objective 40.
Furthermore,
objective 40 preferably is designed such that any stray light from sample 74
which is not to form an
in-focus part of the final image is attenuated by stops, irises or geometrical
light trapping means.
As used herein the terms "geometrical light trapping" and "geometric surfaced"
are intended to
comprise any surface with low reflectance in the wavelength ranges of interest
and which have
surfaces geometrically arranged to direct whatever small amount of light
impinges on them toward
other geometrical light trapping surfaces or "safe" areas where the light will
not degrade operation
or contrast formation of the optical instrument. Thus, substantially most or
all of the light being
trapped or attenuated is absorbed during reflections from successive surfaces
of the geometrical
surface. In some cases only one, or a few bounces, is required to sufficiently
attenuate the light
while in other cases a large number of bounces is required to achieve the
desired level of
attenuation. These geometrical surfaces are similar in theory to the absorbing
surfaces of an
acoustic anechoic chamber or the anti-radar surfaces of a stealth aircraft.
The use of an aperture 90 or an adjustable iris (not shown) in the same
location, is
desirable to exactly match the illumination beam to the numerical aperture of
objective 40 to ensure
that the best possible darkfield image is obtained.
It is presently preferred to employ an adjustable, rather than fixed, iris in
microscope
objective 40 as the opening of such an adjustable iris to its full NA in a
high power objective with
an NA greater than 1.25, when used with a darkfield condenser of NA 1.25 inner
cone, is to allow
objective 40 to operate at an aperture greater than the inner illuminating
cone of condenser 38. In
this configuration, microscope 10 can be employed in an unusual brightfield
mode which
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accentuates surface topography of sample 74 while substantially maintaining a
high contrast and
resolution of the obtained image. When inverted, this image appears to
resemble a conventional
SEM image of a surface. Further, a slight enhancement of the resolving power
of objective 40 can
be obtained due to the increase in numerical aperture. This brightfield mode
can provide a novel
image appearance to provide image information that was previously
unobtainable.
If objective 40 is an infinity corrected objective, then a suitable matching
tube lens
(not shown) is employed to convert the infinity light to fixed focal length
light. It is advantageous
to employ a tube lens with the shortest possible focal length in order to keep
the length of a
coupling tube between the tube lens and the imaging means to a minimum. This
short coupler
length helps to reduce weight and vibration of the imaging means relative to
the object and, or the
tube lens.
Normally the objective 40, or the tube lens in an infinity corrected system,
produces
a primary image with a circular diameter of approximately 20 to 25 mm. Since
the IDC microscope
uses an imaging means generally in the first image plane of the objective or
tube lens, it is often the
case that the imaging means has an active area of only 8 to 12 mm on a side,
in a square or
rectangular format. In this case, the light path carrying the image must be
apertured or attenuated
with geometrical light stop surfaces to eliminate the light outside the active
image area, so that the
light falling outside the active image area does not become stray light in the
system.
If a fluorescence capability is to be provided for microscope 10 and an
infinity
corrected objective 40 and matching tube lens is employed in microscope 10, an
emission filter (not
shown) or filters (such as conventional filter cube set with emission,
excitation and beamsplitter
elements such as are commonly used in reflected light microscopes and which
can also be
employed herein with a standard system of light source and optics for
reflected fluorescent light
microscopy) can be located between objective 40 and the tube lens in the
infinity space. If a fixed
focal length objective 40 is employed, then the emission filter or filters for
fluorescence microscopy
can be included in head 82 of microscope 10.
Where this emission filter (or filters) is used with fixed focal length
objectives, it is
preferred to coat the emission filter onto the thinnest possible filter
substrate so that the deviations
of the image due to the index of refraction of the filter coatings and
substrate will be as small as
possible. The filter or filters can be on a slide or can be on a turret or
filter wheel arrangement, as
will be apparent to those of skill in the art. Where these filters are used to
create artificial color,
when a monochrome digital camera is employed or where they are used for
multiple fluorescence
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techniques or when numerically processed pseudo-color computer driven imaging
is employed,
then the filter turret or wheel can be digitally controlled and electrically
driven.
A fixed focal length objective can be expected to produce a brighter (more
photon
efficient) and a more highly corrected image in the first image plane than an
infinity corrected
system, due to the lower number of surfaces and components relative to an
infinity focused
objective. Where fixed focal length objectives are employed, it can be
desirable to design the
objectives with a much shorter back focal length in order to substantially
reduce the overall height
of the microscope as described above for infinity corrected tube lens to
imaging means coupling.
Microscope 10 can include a single objective 40, or can include two or more
objectives 40 which can be selected for use as desired. In this latter case,
the objectives 40 can be
mounted on any appropriate mounting means, such as the conventional revolving
nosepiece used in
many microscope designs.
The light leaving objective 40 passes through a first aperture 94 and then,
when
leaving head 82 of microscope 10, through a carefully controlled second
aperture 96 which blocks
any light rays not in the desired image forming beam. The walls of head 82 and
a coupler 98 are of
relatively large internal diameter to further reduce stray light and improve
image contrast. The
inner surfaces of head 82 and coupler 98 can also be machined with geometrical
surfaces to control
and substantially eliminate light reflections from reaching the imaging means,
discussed below.
The inner surfaces of the objective 40, head 82 and coupler 98 are preferably
finished in a flat black or other suitable finish to obtain the lowest
possible coefficient of reflection
for light of the wavelengths being employed to form the final image. Generally
this will be flat
black or anodized black finishes.
Before the light containing the image information reaches the imaging means,
it
passes through another aperture or stop 102 which is shaped to further limit
stray light. This
aperture can be a square or other shaped aperture to match the geometry of the
imaging means.
For the present invention, to use most current production objectives, it is
desirable to
add stray light control means to the objectives. These stray light control
means include finishing the
edges of the lens to a fine line surface (for plano-convex and double convex
lens elements),
blacking the outer peripheral surfaces of the lenses and/or finishing the
peripheral surfaces to
geometrical configurations to control stray light bounce, adding apertures or
stops, providing ultra
low reflection or geometrically engineered surfaces on inner diameters of lens
mounts and barrels,
and carefully controlling the antireflection coatings to prevent stray light
from migrating towards
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13
the imaging means.
Objectives for use in IDC microscopes in accordance with the present invention
should also be rigorously checked to determine how closely they accomplish the
theoretical limits
for an objective of their design and for the physical limit, according to
physics, for such an
objective design. Components of the objective should then be adjusted to
achieve the highest
possible conformance with theoretical performance possibilities. The use of a
suitable test slide,
such as that sold under the name "Richardson Test Slide" by Bio-Microtech
Inc., P.O. Box 23,
Bolton, Ontario, L7E 5T1, and described in Richardson, T. (1998), Test Slides:
Diatoms to
Divisions- What are you looking at? Part 1, is very useful in confirming the
performance of each
aspect of the IDC system, including the illuminating, objective and imaging
components and all the
connecting components in or adjacent to the light path.
Traditional eyepieces, whether part of a monocular, binocular or trinocular
design,
have been eliminated in this design to improve the photon efficiency of the
system and to remove
the need to correct the optics in the ocular system to the same high standards
as the rest of the
optical system. Removing the ocular system also reduces costs materially,
provides a lighter, more
compact design, and offers the operator a more user-friendly interface with
less fatigue and virtually
infinite ergonomic flexibility, as the final image is viewed on a video or
digital monitor which can
positioned virtually anywhere to suit the ergonomics of the situation.
In the embodiment shown in Figure 1, the imaging means 106 is a three detector
CCD camera such as a model GP-US532 manufactured by Panasonic, with an
internal prism 110
and three charge coupled array detectors 114, 118 and 122 is placed at the
primary image plane of
the objective (or the objective tube lens combination in the case of infinity
corrected optical
systems). Placing imaging means 106 in the first focal plane of the objective
is presently believed
to be advantageous since it improves the image brightness as otherwise, the
presence of any
intervening optics would introduce light losses, and since it maintains the
highest possible image
resolution and contrast which would otherwise be degraded by any other
intervening optics.
A low light broadcast grade video camera, such a model WV-E590 manufactured by
Panasonic, can also be used as the imaging means. This type of camera is
particularly suited for
low light work where photon damage to the sample must be kept to a minimum. It
is also suitable
for fluorescent work where the image has low light levels and where excitation
energy must be kept
to a minimum to reduce photon damage and bleaching of the sample and
fluorophores.
The electronic image acquired by imaging means 106 is provided to a control
unit
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128 which can contain automatic gain controls, white balance, black balance
and auto iris functions,
all of which can be controlled or limited by the operator of the system for
maximum imaging
control and flexibility.
A particular advantage of this system is the use of a video camera with the
ability to
display fields of image versus frames. When only the odd or even fields are
displayed and the
interlace information is interpolated from the adjacent fields then effective
frame speeds equal to
the field rate can be achieved. This is important when it is desired to study
very high speed motion
with a minimum of motion during the image acquisition time. It is also useful
to use electronic
shuttering to limit the motion during a field duration.
The electronic signal from imaging means 106 and control unit 128 is then
provided
to image inverter system 132 which electronically converts it to obtain either
the luminance,
chrominance, or luminance and chrominance, negative image of the image
provided to it from
control unit 128. A trivial example of the function of image inverter system
132 would be that an
image of a black spot on a white background is converted to a white spot on a
black background
when both luminance and chrominance are inverted. Both control unit 128 and
the image inverter
system 132 can be included as internal or integral components of the imaging
means 106.
Alternatively, internal or switchable programming of control unit 128 can be
used to
accomplish the image inversion. This is especially useful when it is desired
to have the microscope
operate at all times in the inverted mode where one or both of the luminance
or chrominance
information is inverted.
Depending on how the chrominance information is to be handled by the image
inverter system 132, then the resulting color in the final image leaving image
inverter system 132
can either be a color correct image or a color negative image of the image
provided to system 128.
Further, depending on what type of images are coming from sample 74, it can be
desirable to view
the image without the inverting conversion being performed. Accordingly, image
inverter system
132 can forward both the inverted image and the non-inverted image. In this
way, image inverter
system 132 provides as many as four modes of output images. The first mode
being the normal
positive image, the second mode being the negative image with color in the
negative (when
luminance and chrominance are inverted), the third mode being where color is
not in the negative,
but the brightness is the negative (when only luminance is inverted, which can
be especially useful
when the system is used to view samples with stains, such as vital stains, or
known color) and the
fourth mode being where color is negative but brightness is not negative
(which can be useful for
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studying color differences which are accentuated when inverted due to the
characteristics of the
human eye or the camera or the video monitor).
In the embodiment of Figure 1, image inverter system 132 does nothing to alter
the
resolution or the contrast of the image. The resolution and the contrast
information is solely
5 derived from the optical methods employed being darkfield illumination,
optimal correction of the
optics to provide superior image quality and photon efficiency, vibration
control and attenuation,
and careful attention to photon budgets to account for substantially all the
photons leaving light
source 14 to ensure that they contribute to the final in-focus image. Control
of stray light in
microscope 10 is an important factor in obtaining the final high contrast
image.
10 The human visual system is far better adapted to, and used to, processing
information, when image information is presented as black or color on a
substantially white
background, much as text is normally displayed on paper. The darkfield image
is not visually
familiar to the normal viewer and therefore the brain has difficulty
extracting the most information
f'rom the image. A good example is the difficulty we have trying to interpret
or understand image
15 information when viewing a photographic negative, either of a color or
black and white scene.
When we look at a positive image of the same negative we can easily interpret
the information
"correctly", even though both the positive and negative images contain the
same information and
one is only the luminance and chrominance inverse of the other. It has been
found that the image
produced from the IDC system generally need not be enhanced by digital image
processing
techniques as it appears to be located in the region of information which is
best interpreted by the
human mind. The only adjustments that have been found to often be helpful are
adjustments of the
black and white offset levels on the analog or digital control system to
delivery more information
on faint features.
In normal use, the illumination control which provides linear adjustment of
the light
level from zero to the maximum that light source 14 is capable of, is used in
conjunction with the
electronic gain controls of imaging means 106 to deliver the best image for
the information desired.
To study fine details in a sample, high levels of illumination are used and
low electronic gain is
employed, so that electronic noise is minimized and fine resolution is
maximized. For long term
studies at lower resolution, the light level is kept at the lowest possible
level and the electronic gain
is switched to the highest possible setting so that a useable image is
delivered at a low light level,
from the point of view of the sample. For high sensitivity detection of small
particles, background
organisms or structures, both the light level and the gain are set to their
maximum which produces
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strong contrast in fine structures and high optical amplification to that
small structures take on a
high contrast and electronically noisy, and thus easily distinguished,
appearance.
Alternately image inverter system 132 can be implemented in a computer-based
image processing system (not shown) wherein the imaging means is an analog
camera and the
computer contains an image capture card for converting the analog image to a
digital image or
where the imaging means is a digital camera and the computer processes the
digital data directly.
The advantage of using the computer-based image processing system with this
type of microscopy
is that color can be mapped to the acquired image in a defined way to best
suit the application.
Contrast expansion and pseudo-color techniques, along with other known image
processing
techniques, such as edge enhancement, can be beneficial to extract further
information from the
images obtained.
The final acquired and processed image is displayed to the operator on a
monitor 136
which can be an analog monitor or a computer monitor. It can be desirable to
provide switches
such as 140 and 144 to select the video mode being viewed. In the embodiment
of Figure 1, switch
140 selects the positive video image provided via connection 148 and switch
144 selects the
negative video image provided via connection 152.
The final image can be recorded using analog means such as on video tape or
digitally as digital video or digital image files of either still (such as
TIFF, or JPEG type files) or
motion video (such as MPEG, MPEG2 or AVI type files). Current IDC systems are
using S-VHS
format recorders, or RGB professional video recorders. It is contemplated that
DVD or a Raid
Array or similar digital recording strategies will be offered as the recording
means as they become
commercially available and more cost effective. "Casino" type time lapse video
recorders are also
currently used with the IDC to allow studies of samples over long time
interval of hours to days.
Currently, as shown in Figure 2, digital capture from the imaging means 106
whose
S-Video signal 300 is supplied to image inverter system 132 which contains
luminance inverter 301
and chrominance inverter 302 and luminance invert/non-invert switch 303 and
chrominance
invert/non-invert switch 304. The signal from the image inverter system 132 is
supplied to an S-
Video recorder 305 and the signal loops through to a time lapse recorder 306,
monitor 136 and
image capture system incorporated into computer 307. The image capture systems
supplied
currently with IDC systems include IOMEGA Buzz image capture systems or MATROX
Genesis
image capture system in conjunction with a suitable PC computer system. The
Buzz offers a low
cost method for general purpose capture of images. The Genesis is a
professional grade image
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capture system which allows more detail to be collected.
This method of microscopy can be used with high power high numerical aperture
objectives or with lower power objectives. The main limiting factor is the
numerical aperture of the
objective so that the objective aperture is smaller than the inner
illuminating cone of the
illumination system. This makes microscopes in accordance with the present
invention ideal for
examining cells, such as human biopsy or plant cells, at low magnifications
initially and then
switching to higher magnifications later on for more detailed analysis.
As will be apparent to those of skill in the art, it is possible to employ
electrochemical, electroluminescent, fluorescent, liquid crystal or image
intensifier based schemes
to provide the positive to negative conversion in this method. If such means
are used, then
conventional binoculars or trinoculars can be used to view the image of the
object but some of the
contrast and resolution will be lost.
It is further contemplated that ultrafine focussing of the microscope can be
accomplished by controlled distortion of the vibration control braces 78 of
microscope 10. For
example, if a hydraulic cylinder (not shown) is used to couple the braces 78
together, then adding
fluid to the hydraulic cylinder will force the braces 78 apart and deflect
objective 40 towards
sample 74 very slightly, thus providing a very fine focus control. If a very
fine screw (not shown)
is used to drive a very small bore piston (not shown) into a cylinder (not
shown) filled with
hydraulic oil and the resulting pressurized oil is supplied to the hydraulic
cylinder connecting
braces 78, then a very ultrafine focus can be implemented. Such an adjustment
screw can be under
computer or external electrical control. The same type of function can be
accomplished with a
screw mechanism either in tension or compression between the braces 78 so that
adjustment of the
screw mechanism accomplishes the fine focus.
It is also contemplated that microscope 10 can employ one or more
piezoelectric
struts (not shown) between braces 78 to accomplish the ultrafine focussing of
the microscope.
Variation of the voltage on the piezoelectric struts will shift the focus of
the microscope slightly.
Alternatively, the struts can be fabricated in two units and can include a
piezoelectric layer
sandwiched between the upper and lower halves of the braces so that the piezo
element can vary its
thickness and by so doing alter the length of the braces.
Figure 3 shows such a configuration of two braces containing piezoelectric
elements
for active Z position control. The top portion of the body of the microscope
82 is connected to the
top halves of each brace 202. The lower surface of brace 202 is adhered to an
insulating and
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conductive top electrode 203, which allows the connection of the piezo to a
voltage source.
Electrode 203 is connected to piezo electric element 204 which is adhered to
the bottom electrode
205. Electrode 205 is adhered to the bottom half of the brace 201. A voltage
applied between
electrodes 203 and 205 causes a change in dimension of piezo element 204 and
moves the imaging
means and related optics relative to the sample object 74.A further
application of this piezoelectric
system is to move the microscope Z adjustment in synchrony with a vibrating
sample 74 to obtain
images of samples undergoing or exhibiting fixed frequency vibrations which
have apparently
halted the motion of the sample, at least in the Z plane. Alternately a three
axis piezo mount can be
used to secure the objective to the microscope body. By driving this mount in
synchronized three
dimensional patterns it can be possible to freeze the motion of a sample
object in rapid oscillation
by matching the motion of the objective to the motion of the object.
It is possible that reflections from the surface of the CCD camera or other
imaging
means can bounce back and forth in the space between the CCD camera and the
back lens of
objective 40 or the tube lens. It is contemplated that a benefit can be
obtained by inserting a
photonic valve, such as a one way mirror (not shown), to eliminate light
returning to the objective
lens from the imaging means and thereby eliminate a possible source of stray
light and to improve
the contrast.
The overall size of microscope 10 can be substantially reduced by
incorporating the
light source 14 into the internal housing of the condenser 38. Such a design
was proposed by Zeiss
in the 1930 for darkfield condensers. If a brightfield and darkfield
combination condenser such as
the Barnard coaxial, described above, is employed then the size can be kept
very small while
maintaining both brightfield and IDC modes of operation.
In order to provide a low power and extremely robust and compact IDC
microscope
for field use in rough environments, one or more light emitting diodes (not
shown) can be employed
as the light source and a conical prism-type condenser can be employed to take
best advantage of
this type of illumination.
Where color correction is an important factor, an array of light emitting
diodes of
different wavelengths can be used with an LED control means to vary the
relative brightness from
each of the LED's. In this way a color matched lighting system can be
obtained. Depending on
how the LED's are arranged, the color and the position and style of
illumination can be varied to
meet the needs of the application.
Figure 4 shows a cutaway of the internal construction of an objective for an
IDC
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19
microscope. Light enters the objective 40 through the first lens 400, here
shown as an oil
immersion lens, stray light or high numerical aperture light which is not to
form part of the in focus
image as determined using photon budgeting techniques, is incident on the
lower surface of mount
408 for lens 405. In focus light passes through lens 405. The geometrical
light trapping features of
mount 408 are located on the top 416 and bottom 412 surface of the mount 408.
Detail 460 shows
that the features 412 and 416 are not symmetrical but instead have surfaces
444 and 448 on the
lower surface which are designed to reflect incoming light 480 away from the
optical axis as
reflected light 470 and into geometrical surface 404 on the inside of the
objective mount or casing.
Where the outer edge of a lens 405 is flat then the outer edge can be painted
flat black to reduce
reflections and stray light. Where the edge of the lens allows as in double
convex lens 424 and
plano-concave lens 432 the outer edge is polished to a fine point or surface
so that internal
reflections or diffusion are minimized. Stray light in the upper section of
the objective is controlled
by geometrical light trapping surfaces 416 on the lens mounts 408. The upper
lenses are mounted
in component 421 with lenses 424 and 432 and geometrical light trapping
surfaces 420, 422, and
428. Stray light is prevented from leaving the objective by aperture stop 440
and its inner
geometrical light control surface 436 working in conjunction with surface 437.
Reflections from
the tube lens or the imaging means are further controlled by geometrical light
trapping surface 436.
The above-described embodiments of the invention are intended to be examples
of
the present invention and alterations and modifications may be effected
thereto, by those of skill in
the art, without departing from the scope of the invention which is defined
solely by the claims
appended hereto.
