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Description  |
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The present invention relates to a method and device for producing
phase-contrast images with a microscope which scans the object
point-by-point.
BACKGROUND
Optical scanning microscopes, frequently also referred to as "laser scan
microscopes", have been known for a long time. For example, the "Journal
of Microscopy", Vol. 138, Pt 1, (Apr. 1985on pages 29-34, describes an
optical scanning microscope of confocal construction which effects the
scanning of the object by means of a moving table. The optical system of
this microscope is stationary and is designed to focus the smallest
possible light spot which can be produced with limited diffraction in the
object plane. A condenser, in the form of a second objective, serves to
collect the light which passes through the object. A photomultiplier is
positioned behind said objective (after corresponding deflection of the
beam), and the signals of the photomultiplier are used to create the
object image on a monitor.
A scanning optical microscope of a somewhat different construction is shown
in the Carl Zeiss publication W41-910e, "Laser Scan Microscope: An Optical
Scanning Microscope", printing annotation IX/84. In this scanning
microscope, the laser beam, which is used for the scanning, is itself
moved over the stationary object. The construction of this scanning
optical microscope is shown in FIG. 1 and will be described further below
with reference to said figure.
In conventional microscopes, "Zernicke phase contrast" is a contrast method
which has been used for a long time. The theory and practice of this
method are, for example, described in great detail in the book by Dr. H.
Beyer, Theorie und Praxis des Phasenkontrastsverfahrens [Theory and
Practice of the Phase Contrast Method], published by Akademische
Verlagsgesellschaft of Frankfurt/Main in 1965. Briefly stated, the method
consists in imaging a diaphragm aperture, of annular or other preselected
geometric shape in the rear focal plane of the condenser of a
transmitted-light microscope, on a corresponding annular phase plate in
the pupil of the objective employed. The phase-contrast image is produced
by interference of the light which passes directly through the object and
the phase plate with the light which is diffracted on the object and
passes through the objective alongside the phase annulus. A condensed
description of this effect can also be found in ABC der Optik, editor Karl
Mutze, published by Verlag Werner Dausien of Hanau/Main in 1961, on pages
634 to 638.
In their book Theory and Practice of Scanning Optical Microscopy, Academic
Press 1984, T. Wilson and C. Sheppard describe how the Zernicke phase
contrast method can also be applied to optical scanning microscopes. This
reference utilizes a microscope design which is well-known in conventional
microscopy, namely, the design uses a condenser and an annular diaphragm
aperture in its rear focal plane. The above-described prior art optical
scanning microscope for producing phase-contrast images, shown in the Carl
Zeiss publication W41-910d, also uses this same conventional construction.
Copending U.S. patent application Ser. No. 30755 discloses an X-ray
microscope which is suitable for producing phase-contrast images. This
X-ray microscope has a condenser in the form of a first, so-called zone
plate for the irradiating of the object and an objective in the form of a
further zone plate with which the object is imaged. A phase-shifting
element, which effects the phase contrast in a manner similar to that used
in conventional optical microscopy, is arranged on the Fourier plane of
the second zone plate.
Scanning microscopes for radiography are also known. In these apparatus,
the object is moved in the form of a raster under an X-ray beam which is
focused in the form of a spot, and the X-radiation transmitted through the
object is directly detected by a detector. As a rule, no condenser is
included in X-ray scanning microscopes because the zone plates which would
be used as condensers have only very slight diffraction efficiency. Due to
the elimination of the condenser, such microscopes can therefore operate
with a lower X-ray dosage, i.e., radiation which is gentler on the object.
However, no device for producing phase-contrast images is yet known for
scanning X-ray microscopes, since heretofore it has been assumed that a
condenser is required for such a method and, accordingly, that it would be
necessary to use a higher and undesirably damaging X-ray dose.
It is the object of this invention to create a phase-contrasting method and
apparatus which can be used with scanning microscopes to produce
comparable phase-contrast images at less expense and with lower radiation
intensities.
SUMMARY OF THE INVENTION
The invention makes use of the surprising discovery that phase-contrast
images can be produced in scanning microscopes without requiring the use
of a condenser or any other radiation-collecting systems intervening
between the object plane and the radiation-sensitive detector. The
detector can be appropriately positioned directly below the object plane,
and its radiation-sensitive area is adapted to encompass the radiation
passing through the phase-shifting regions in the microscope's objective
which directs the illuminating radiation onto the object. This adaptation
is accomplished either by placing an apertured diaphragm in front of the
detector or by designing the detector so that its photosensitive area
matches the shape of the phase-shifting regions.
It has been found that this novel design, without any condenser between the
object and the detector, is suitable for apparatus which operate with beam
scanning, i.e., which guide the illuminating radiation over the object to
be imaged, and is similarly suitable for apparatus with which the scanning
movement is achieved by means which carry the object stage in raster-like
movement. The precise position of the apertured diaphragm and the detector
relative to the object plane is not critical as long as the radii of the
diaphragm apertures are adapted to encompass the cone of radiation which
is defined by the phase plate and the objective. This adaptation can be
calculated or established experimentally.
The geometric relationships are, to be sure, dependent on the aperture and
the linear magnification of the objective used; but solutions can always
be found for different objectives, either by placing the diaphragm or
annular detector surfaces at different distances from the object or by
placing several diaphragms of different annular diameter and different
annular width, for example, on a turret.
The invention thus makes it possible to dispense entirely with a condenser
for producing phase contrast in scanning microscopes, without any loss in
the quality of the image.
Since the radiation to be detected is not attenuated by absorption in a
condenser or other radiation-collecting systems, one can operate with
lower radiation intensity.
The method is therefore not only suitable for optical microscopes which
operate in the visible spectrum but, also, is particularly suitable for
X-ray microscopy since, as indicated above, it is very difficult to
produce condensers having sufficiently high X-ray transmission
characteristics.
Further advantages of the invention can be noted from the following
detailed description in which reference is made to the accompanying
drawings.
DRAWINGS
FIG. 1 is a basic diagram of an optical scanning microscope which is known
per se and operates with beam scanning.
FIG. 2 is a schematic representation showing the essential optical
components for producing a phase-contrast image in an optical scanning
microscope with stage-scanning.
FIG. 3 is a schematic representation showing the essential optical
components for producing a phase-contrast image in an optical scanning
microscope with beam-scanning.
FIG. 4 shows, in an enlarged schematic top view, a detector which can be
used as an alternative to the detector (36) or (136) in FIG. 2 or FIG. 3
respectively.
FIG. 5 is a schematic representation similar to FIG. 2 showing the optical
components for producing a phase-contrast image in a scanning optical
microscope similar to that shown in FIG. 2 but using an objective of
shorter focal length.
FIG. 6 shows the image of an object in phase contrast, taken by the
conventional technique with a condenser.
FIG. 7 shows the object of FIG. 6, taken with the construction outlined in
FIG. 3.
FIG. 8 shows the same object as in FIG. 7 in defocused condition.
FIG. 9 shows schematically the essential optical components for producing a
phase-contrast image in an X-ray scanning microscope.
DETAILED DESCRIPTION
FIG. 1 shows the optical scanning microscope known from the aforementioned
Carl Zeiss publication W41-910e. This prior art apparatus is constructed
as follows:
The beam of illuminating radiation, for example, from an argon laser (1) is
widened by a telescope lens system (3, 4) after deflection on a mirror (2)
and fed to a scanning system (8) consisting of two mirrors which can be
swung perpendicular to each other. The beam of light cyclically deflected
by said mirrors is then reflected into the observation ray path of a
microscope by a beam splitter (11) and focused on the specimen (13) by an
objective (12). A lens (9) serves to image the scanning system (8) in the
objective (12). A lamp (22) and a collector (23) comprise an auxiliary
illuminating system which, by means of another beam splitter (10), is
coaxially superimposed on the beam of the laser (1). This makes
conventional microscopic observation of the object (13) possible via the
observation ray path, which is shown schematically as a tube lens (24), a
reflecting prism (25), and an eyepiece (26).
For the display of the object (13) by a scanning microscope, a first
detector (7) is provided in the incident light ray path, the detector
being arranged behind a lens (29) and a filter (6), to monitor the
radiation emitted by the specimen and collected by the objective (12)
after its return via the deflection unit (8). The detector (7) is located
in a partial-ray path which is divided out by the beam splitter (5)
between the enlargement lens system (3, 4) and the scanning device (8).
A second detector (27) is provided in order to detect, in transmitted
light, the light scattered forward in the direction of the beam by the
specimen (13). The second detector (27) is also arranged in a partial-ray
path behind a lens (19), the partial-ray path being divided out by a beam
splitter (18) from the transmitted light auxiliary illuminating ray path
of the microscope. The ray path of the transmitted light auxiliary
illumination consists of a lamp (21), a collector (20) of a lens (17), a
reflecting mirror (16), and a condenser (14) below the specimen (13).
The signals of the two detectors (7) and (27) are fed to a monitor (28)
with attached image storage. The monitor is synchronized with the tilt
mirrors of the scanning device (8) and can also selectively display either
an incident-light or a transmitted-light image.
For the displaying of phase-contrast images in transmitted light, the
objective (12) includes a phase-shifting element of preselected geometry,
e.g., annular, and the condenser (14) of this known optical scanning
microscope, similar to the condenser in a conventional microscope, has
corresponding annular diaphragm apertures in its rear focal plane.
In accordance with the invention disclosed herein, it has been found that
it is possible to dispense with condenser (14) and the lens system (15-19)
arranged behind it. To explain this more fully, reference is now made to
FIGS. 2 and 3. FIG. 2 shows the phase-contrast objective of an optical
scanning microscope, depicting it schematically as lens (31) and phase
plate (32), with the phase-shifting elements of this phase-contrast
objective being shown as phase annulus (33). It will be appreciated that
the phase annulus (33) is normally vapor-deposited directly on the lens of
the objective instead of on an additional glass plate as shown in this
simplified schematic.
An annular diaphragm (35) is arranged at a distance (a) behind the object
plane (34) in which the objective (31) focuses the scanning light beam
with limited diffraction, and directly behind said annular diaphragm there
is arranged a photoelectric detector (36) having a relatively large
photosensitive area. The detector can, for instance, be a diode of the
"PIN 10 D" type of the UDT Company. This detector diode has a
photosensitive area of a diameter of 11.3 mm.
The cone of rays (37) passing through the phase annulus (33) is shown
shaded. The distance (a), between the diaphragm (35) and the object plane
(34), and the diameter (c) of the annular diaphragm aperture (38) have
been so selected that this cone of rays (37) passes through the diaphragm
aperture (38) and strikes the photosensitive area of detector diode (36).
With this novel and simple construction, it is possible to obtain
phase-contrast images by moving the object being examined in raster-like
manner in the object plane (34) and utilizing the signals of the detector
(36) for the imaging. The position of the annular diaphragm (35) and the
detector (36) relative to the object plane is not critical as long as,
when preselecting distance (a) and diameter (c), the cone of rays (37)
defined by the phase plate (32) and the objective (31) passes through the
diaphragm aperture (38). An interference filter may be arranged above or
below the diaphragm (35) for the suppression of interfering ambient light.
The invention disclosed herein can also be used to produce phase-contrast
images when the object itself is not moved but, instead, the laser beam is
scanned over the stationary object. This situation is shown in FIG. 3. The
same components have been used in FIG. 3 as in the embodiment of FIG. 2.
However, during the scanning of the beam, a partial trimming of the cone
of rays (137) passing through the phase annulus (33) occurs due to the
movement of the laser beam [indicated by the double arrow (A)]. However,
in spite of this trimming, no detrimental effect can be noted on the
quality of a phase-contrast image produced in this manner. This can best
be explained by the following example: When using an objective like the
Carl Zeiss Planapochromat 40/0.95 PH3 type, the cone of rays (137) on the
detector varies by less than 0.1 mm around the central position during the
scanning; and since the width of the annular diaphragm aperture (38) used
in this example is two millimeters, the slight portion of trimmed light is
of no consequence.
It is obvious that the diameter (c) or the distance away (a) of the
diaphragm (35) must be changed when an objective with a different aperture
or with different dimensions of the phase annulus is to be used. This is
shown in FIG. 5. Here a detector (46) has a diaphragm (45) arranged in
front of it, and the latter has a diaphragm aperture (48) of the same
diameter (c) as the aperture (38) in FIG. 2. However, in this case the
detector (46) and diaphragm (45) are located at a distance (b) below the
object plane (44). Distance (b) must be smaller than distance (a) in FIGS.
3 and 4 in order to assure that aperture (48) encompasses the cone of rays
passing through the phase annulus (43), because these rays are focused by
an objective (41) of larger aperture and smaller focal length. It can be
appreciated from these just described geometric considerations that it
would be preferable to effect such required adaptation by appropriately
altering only the diameter and width of the annular diaphragm aperture
(48) while maintaining a constant distance between the diaphragm and the
object plane. To accomplish this purpose, it would be possible, for
example, to arrange several diaphragms of varying dimensions on a turret.
It should also be noted that it is possible to dispense entirely with a
diaphragm arranged in front of the photodetector by using, as shown in
FIG. 4, a detector (40) having its photosensitive area (39) already shaped
to encompass the phase-shifted radiation. Such detectors are, for example,
available from the EG & G Reticon under the designation "Circular Array
RO0720B" or "Self-Scanned Circular Photodiode Array RO0064N". Since these
detectors consist of several individual diodes in annular arrangement,
they can be utilized for creating phase-contrast images not only from the
radiation integrally incident on the entire circular area but also from
that incident only on preselectable angular areas. This makes possible
additional advantageous contrasting methods similar to so-called
monolateral oblique illumination, for example.
FIGS. 6-8 permit comparison of phase-contrast images which were recorded in
accordance with the state-of- the-art and those recorded in accordance
with the present invention. A section through a young trout (cut
crosswise) served as object. The picture of FIG. 6 was taken with a Carl
Zeiss laser scan microscope of the type described with reference to FIG.
1, having a Planapochromat (40/0.95) objective and a condenser, the turret
of which was set to phase annulus "No. 3". The electronic zoom of the
laser scan microscope was set at a magnification factor of 45 x so that
the linear magnification amounted to a total of about 1350 x.
The picture of FIG. 7 was taken on the same microscope with the same
setting but with the apparatus being modified according to the invention
herein, namely: (a) the condenser (14) was removed; (b) the
photomultiplier (27) of FIG. 1 was disconnected; and (c) the components
shown in FIG. 2 or FIG. 3 were positioned below specimen (13), with the
above-mentioned diode "PIN 10 D" of the UDT Company used as the detector
for photographing the phase-contrast image. As is apparent from a
comparison of FIGS. 6 and 7, the invention produced a phase-contrast image
of equally good quality as was produced with the more complex and more
expensive prior art apparatus.
Defocusing was thereupon effected out of the object plane. The resulting
picture is shown in FIG. 8. As can be noted, a contrast reversal takes
place in the image, in the same manner as it does in the phase contrast of
conventional microscopy.
In the X-ray scanning microscope shown in FIG. 9, a zone plate (101) serves
for the punctiform focusing on the object (104) of radiation (110) emitted
by a source of X-rays (not shown). Such a zone plate is for example
described in the above-mentioned U.S. patent application Ser. No. 130775.
The object (104) is located on a mechanical stage which is moved in
raster-like manner by a well-known device which is not shown.
Seen in the direction of the light, a phase plate (102) is arranged in
front of the zone plate (101) in the focusing plane of the zone plate
(101), i.e., in the pupil plane of the objective. This phase plate bears a
phase annulus designated (103) which shifts the phase of the part of the
X-radiation passing through it with respect to the part of the radiation
passing outside the annulus (103). The phase shift is preferably set at 90
or at some other amount selected with a view toward the best possible
image contrast.
Behind the object plane (104) in which the objective (101) focuses the
scanning X-ray beam with limited diffraction, there is arranged an annular
diaphragm (105) at a distance away (a) and, directly behind it, a detector
(106) having a relatively large photosensitive area. The detector can, for
instance, be a proportional counter such as described in "Proceedings of
SPIE--The International Society for Optical Engineering", Vol. 733 (1986),
under the title "Soft X-ray Optics and Technology", on pages 496-503.
The cone of rays (107) passing through the phase annulus (103) is shown
shaded. The distance (a) between the diaphragm (105) and the object plane
(104) and the diameter (c) of the annular diaphragm aperture (108) have
been so selected that this cone of rays (107) passes through the diaphragm
aperture (108) and strikes the detector (106) within the photosensitive
area.
It is now possible to obtain phase-contrast images by moving the microscope
object to be examined in raster-like manner in the object plane (104) and
utilizing the signals of the detector (106) for the imaging. The position
of the annular diaphragm (105) and the detector (106) relative to the
object plane is not critical as long as distance (a) and diameter (c) are
suitably selected so that the cone of rays (107) which is defined by phase
plate (103) and objective (101) passes through the diaphragm aperture
(108). In the manner noted above, an interference filter can be arranged
above or below the diaphragm (105) in order to suppress interfering
ambient light.
In addition to the phase annulus (103), the phase plate (102) also bears a
central diaphragm (109). This diaphragm serves to keep that part of the
X-radiation which otherwise passes unaffected through the phase plate
(102) away from the object (104). This is done to protect the object (104)
from unnecessary radiation. This is, due to the limited X-ray diffraction
properties of the object (104), this direct radiation would not be
diffracted sufficiently to pass through the diaphragm aperture (108) and
so would not interfere there with the phase-shifted part of the
X-radiation. Therefore, such direct radiation would not enhance the image
but rather would only unnecessarily act on the object (104).
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