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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an image formation-type soft X-ray
microscopic apparatus with high resolving power which is mainly used for
observing organisms.
2. Related Background Art
X-ray microscopes hitherto proposed are roughly divided into the following
four types:
(1) A projection enlargement type which has no optical system and in which
a sample is placed at a position near an X-ray point source in the
divergent pencil of the X-rays generated from the X-ray source, and an
X-ray film or a two-dimensional X-ray detector is disposed at a position
behind the sample at a distance therefrom.
(2) An adhesion type which has no optical system and in which an X-ray
source, which supplies a bundle of substantially parallel X-rays, is used,
and a resist is caused to adhere as a sample to the X-ray source. In this
case, a synchrotron radiation source (referred to as "SR" hereinafter), a
plasma X-ray source or an electron beam excitation X-ray source is used as
the X-ray source.
(3) A scanning type in which an X-ray beam is narrowed into a small spot by
an optical system, and the beam and a sample are relatively scanned. In
this case, SR is used as an X-ray source, and a Fresnel zone plate
(referred to as "FZP" hereinafter), a multilayer film mirror or a total
reflection mirror is used as an optical element for narrowing the X-ray
beam into a small spot.
(4) An image formation type in which X-rays are condensed on a sample by
using an X-ray source comprising SR, a plasma X-ray source or an electron
beam excitation X-ray source and an optical element such as FZP, a
multilayer film mirror or a total reflection mirror, and an image of the
sample is formed on a film, a fluorescent plate or a two-dimensional X-ray
detector by using the same optical element.
High-resolution observation of living organisms cannot be easily made by
the above-described conventional X-ray microscopes from the technical
viewpoint because the microscopes are insufficiently optimized and applies
large amounts of X-rays.
Namely, although the projection enlargement type (1) is required to have a
high-luminance X-ray point source, exposure for a long time is required
because of its insufficient intensity, and dynamic observation is thus
difficult. In addition, since the sample must be sliced in order to avoid
a deterioration in resolving power caused by the influence of Fresnel
diffraction, it is difficult to observe a living sample.
Since the adhesion type (2) has no high-resolution detector other than the
resist, the development of the resist is necessary, and real time
observation is thus difficult. In addition, since the magnification is 1,
enlargement observation separately using an electron microscope or the
like is required. Further, destructive observation, in which the sample is
sliced, is required for avoiding a deterioration in resolving power caused
by the influence of Fresnel diffraction in the same way as the projection
enlargement type (1).
The scanning type (3) has the disadvantage that, since an X-ray source
having good directivity is required, a large X-ray source such as SR must
be used, and the size of the apparatus is significantly increased. In
addition, since the scanning time, i.e., the exposure time, is long for
obtaining a desired image, dynamic observation is difficult.
Since the image formation type (4) exhibits a low degree of efficiency when
FZP is used, a large X-ray source such as SR must be used as a
high-intensity X-ray source. In addition, the image formation type (4),
which uses a mirror, has a disadvantage in that the resolving power cannot
be easily improve, and the size of the optical system is increased. This
type is thus insufficiently optimized.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an image formation-type
small X-ray microscopic apparatus which is capable of dynamically
observing a living sample with high resolving power of about 20 nm and
minimum X-ray exposure, without the same being fixed and broken.
The X-ray microscopic apparatus of the present invention is basically of
the above-described image formation type. As shown in FIG. 1, the X rays
emitted from an X-ray source are condensed on a sample by a single concave
aspherical multilayer film mirror condenser, and an enlarged sample image
formed on a two-dimensional X-ray imaging element by using a phase zone
plate PZP as an image formation optical system.
It is effective to use as the single concave aspherical multilayer film
mirror condenser a rotary elliptical multilayer reflecting mirror which
has a most simple shape and which can be easily manufactured.
A plasma X-ray source using a pulse laser is used as a pulse X-ray source,
and laser beams are condensed on a target so that X-rays are generated
from a small region of the target. The target is disposed at the first
focal point of the rotary elliptical multilayer film reflecting mirror,
and the sample is disposed at the second focal point thereof. The
apparatus has a system in which the X-rays are monochromatized by the
multilayer film mirror, and photon counting imaging is performed by using
the X-rays of one pulse generated by excitation from the pulse laser.
Specifically, in the photon counting imaging with one pulse, if the
wavelength of the X-rays is .lambda. and the spectral width is
.DELTA..lambda., the X-ray intensity of the pulse X-ray source is adjusted
so that the maximum number n.sub.man of detected photons incident upon the
two-dimensional X-ray imaging element is within the following range:
25.ltoreq.n.sub.max <.lambda./.DELTA..lambda.
In this case, the pulse width is 1 .mu.s or less, and the pulse X-ray
source used has intensity which allows imaging with one pulse. If the
number of periods N.sub.c of the layer structure of the multilayer film
reflecting mirror is 50 to 400, the X-rays are monochromatized so that the
value of .lambda./.DELTA..lambda. is 50 to 400, and the X-rays emitted
from the pulse layer X-ray source are condensed on the sample by the
rotary elliptical multilayer film reflecting mirror. An enlarged image of
the organism sample is formed by the objective optical system comprising
the phase zone plate having high efficiency and high resolving power.
The wavelength range of the soft X-rays used is 2.3 to 4.4 nm for observing
the interior of the organism sample having a thickness of about 10 .mu.m,
without the sample being fixed and broken. This wavelength range allows
the protein and the lipid in the organism to be recognized as contrast
differences and the sample having a thickness substantially the same as
the cell thickness to transmit the X-rays.
In order to observe a living sample, the apparatus is configured from the
following viewpoints:
(a) Since the path of the X-rays is under vacuum, the sample is observed in
a state wherein it is received in a container which contains water and has
a thickness of about 10 .mu.m.
(b) In order to observe a moving organism, the exposure time is several
.mu. seconds, and a pulse X-ray source and an image formation type of
optical system are employed.
(c) In order to observe a sample with producing minimum radiation damage, a
phase ZP, which can maintain high resolving power of about 20 to 50 nm and
high efficiency, is used as the objective optical system, and the X-rays
are monochromatized by using a condenser optical system comprising a
concave reflecting mirror which is a multilayer film mirror. This permits
the photon counting imaging.
(d) A two dimensional X-ray imaging element is used for real time
observation, and the sample is observed by an optical microscope, which
generally produces little damage to the sample, and, if required, the
sample is observed by an X-ray microscope.
Since X rays significantly damage organisms and easily exceeds the lethal
dose, it is necessary to design the X-ray microscope so that a required
image can be obtained with the minimum dose. The present invention
configured as described above therefore employs an imaging method which
uses a photon counting method. In order to dynamically observe an organism
by using this photon counting imaging method with producing the minimum
damage, the minimum X-ray dose, the detection limit contrast, the
detection limit protein thickness, the gradation of the X-ray image
formed, the dosage (the absorbed X-ray dose per unit mass), the pulse
width, and the spectral width are optimized in view of the following
matter:
(i) Detection Limit and Dose Amount of Photon Counting Imaging
FIG. 2 is a drawing which shows a state of two-dimensional photon counting.
In the drawing, the surface of the sample is divided into small regions
formed by the resolving power .delta. and the focal depth 2D.sub.f, each
of the regions corresponding to one pixel of the imaging element. An image
is formed by differences between the numbers of the photons passing
through the respective pixels. The number of the photons incident on each
of the pixels is n.sub.0, and the numbers of the photons passing through
the pixels are various values depending upon the transmittance.
It is assumed that the detection of the X-ray photons in the adjacent
pixels defined by the resolving power is an independent probability
phenomenon and that the X-ray photons obey a Poisson distribution. (The
influence of MTF, flare and ghost in the optical system is excluded.) It
is also assumed that contrast is mainly formed by differences in
absorption, and that the X-rays, which are lost to the outside of the
imaging optical system by diffraction scattering, are negligible. (A dark
field illumination method utilizing only diffraction scattering X-rays for
forming an image is excluded.)
If the average of the difference in number of the photons of the adjacent
pixels is greater than the dispersion to some extent, it is possible to
detect an image. If the number of the photons applied is n.sub.0 [photon
number/pixel], therefore, the following equation is established between
the average value E and the dispersion of the photons detected in pixels
p.sub.1 and p.sub.2 and the detection limit SN ratio (S/N).sub.d.
##EQU1##
If the transmittance is T, the average is the following:
E(p.sub.1)=T.sub.1
E(P.sub.2)=T.sub.2,
and the dispersion in a Poisson distribution is the following:
V(P.sub.1)=T.sub.1
V(P.sub.2)=T.sub.2
Since the number n.sub.1 of the X-ray photons detected is n.sub.1 =T
.multidot.n.sub.0, as shown in FIG. 2, n.sub.1 and n.sub.2 are the
following:
n.sub.1 =T.sub.1 .multidot.n.sub.0
n.sub.2 =T.sub.2 .multidot.n.sub.0
The contrast C is defined by the following equation:
##EQU2##
From the equations (1) and (2), the following equation is obtained:
C.sup.2 (n.sub.1 +n.sub.2).gtoreq.(S/N).sub.d.sup.2
However, when the detection limit contrast C.sub.d <<1, the maximum number
of the photons detected is expressed by the following equation:
n.sub.max .perspectiveto.(n.sub.1 +n.sub.2)/2
The detection limit contrast C.sub.d is therefore expressed by the
following equation:
##EQU3##
Although the gradation from zero to the maximum photon number is generally
obtained, the reproduced gradation number d.sub.r, which allows the
formation of an image with reliability, is the following:
##EQU4##
The SN ratio at the detection limit of X-ray photons depends upon the
detection method used, the type of the detector used and the like. If it
is assumed that an image can be expressed with gradation by using as
significant information the photon number n, which is divided by
n.+-..DELTA.n/2 (wherein .DELTA.n=2.sqroot.n(.+-..sigma.: standard
deviation)), the photon number at the discrimination limit is expressed by
the following equation:
##EQU5##
When n.sub.max >>.sqroot.n.sub.max >>1, the SN ratio at the detection
limit of the X-ray photons is expressed by the following equation:
##EQU6##
If the photon number is considered as significant information for each
.DELTA.n=4.sqroot.n(+2.sigma.), the following equations are obtained:
##EQU7##
When n.sub.max >>.sqroot.n.sub.max >>1, the SN ratio at the detection
limit of the X-ray photons is expressed by the following equation:
(S/N).sub.d .perspectiveto.2.sqroot.2
If .DELTA.n=6.sqroot.n(+3.sigma.), the following equations are obtained:
##EQU8##
Typical photon numbers for gradation are n=0, 1, 4, 9, . . . in a case of
(S/N).sub.d .apprxeq..sqroot.2, n=0, 4, 16, 36, . . . in a case of
(S/N).sub.d .perspectiveto.2.sqroot.2 and n=0, 9, 36, 81, . . . in a case
of (S/N).sub.d .perspectiveto.3.sqroot.2.
If the thickness of the sample is t, the thickness of protein is t.sub.p,
and the linear absorption coefficients of water W and protein P are
A.sub.w and A.sub.p, as shown in FIG. 3, the X-ray transmittance of water
T.sub.W is expressed by the following equation:
T.sub.W =exp (-A.sub.W .multidot.t) (6)
The X-ray transmittance T.sub.s of the sample is thus expressed by the
following equation:
T.sub.S =T.sub.W .multidot.exp{-(A.sub.P -A.sub.W).multidot.T.sub.p }(7)
Since A.sub.P >A.sub.W within the wavelength region (2.3 to 4.4 nm) of the
water window, the maximum transmittance T.sub.smax of the sample is
expressed by the following equation:
T.sub.smax =T.sub.W
The photon number at the maximum transmittance is the maximum number
n.sub.max of the photons detected.
From the equation (2) and the equations, T.sub.1 =T.sub.W and T.sub.2
=T.sub.S, the thickness t.sub.pd of the protein P at the detection limit
within the focal depth is expressed by the following equation:
##EQU9##
The damage to the organism produced by X-ray irradiation is determined by
the dose amount. (The dose amount is the X-ray absorbed dose per unit
mass.)
If the average transmittance T.sub.sm of the sample is .intg.T.sub.s
dS/.intg.dS, the density is .rho., the resolving power of the microscope
is .delta., the transmittance of the window of the sample container is
T.sub.c, and the objective efficiency is .eta..sub.0, the average
absorption factor is (1-T.sub.sm) and the irradiated photon number is the
following:
n.sub.0 =n.sub.max /(T.sub.w .multidot.T.sub.c .multidot..eta..sub.0)
From the photon energy h.gamma. and the mass .rho..multidot..delta..sup.2
.multidot.t, the dosage D.sub.m (.delta.) is expressed by the following
equation:
##EQU10##
According to the above-described equations, if the maximum number n.sub.max
of the photons detected per pixel is known, the detection limit contrast
C.sub.d and the reproduced gradation number d.sub.r are determined. If the
coefficient of linear absorption A.sub.p, A.sub.w are determined from the
wavelength of X-rays, the thickness t.sub.pd of protein at the detection
limit is determined. Further, from the average transmittance T.sub.sm of
the sample, the transmittance T.sub.c of the window of the sample
container, the transmittance T.sub.w of water, the efficiency .eta..sub.o
of an object, the density .rho. of the sample, the thickness t of the
sample and the resolving power .delta. of the object, the dosage D.sub.m
(.delta.) is determined in accordance with the equation (9).
Namely, in the photon counting imaging method of the present invention, if
the maximum number n.sub.max of the photons detected is known, main
performance, i.e., the detection limit (contrast and gradation) and the
dosage are determined.
(ii) X-Ray Microscopic Imaging with Minimum Exposure
In the present invention, on the basis of the above-described results of
principal analysis, the minimum X-ray exposure optimum for the photon
counting imaging method is determined in accordance with the following
method:
It is suitable for practical use in view of the examples described below
that the maximum number of the photons detected is within the following
range:
25.ltoreq.n.sub.max <200
In accordance with this, the minimum exposure is set so that the maximum
number of the photons detected per pixel is within the above range.
The above-described maximum number of the photons detected is determined on
the basis of the investigation below. The prerequisites are the following:
(1) The wavelength used is about 2.5 nm within the wavelength range (2.3 to
4.4 nm) of the water window which exhibits good transmittance of water and
which permits the contrast of an organism to be easily obtained.
(2) The linear absorption coefficients of water and protein at the
wavelength are the following:
A.sub.w =0.13/.mu.m
A.sub.p =1.5/.mu.m
(3) The thickness t of the sample is 10 .mu.m, which allows the observation
of one cell, in consideration of the balance between the transmittance of
water and the thickness of the cell.
(4) The average transmittance T.sub.sm is determined on the assumption that
the average thickness of cell protein is expressed by the following
equation:
T.sub.pm =0.15.multidot.t
(5) The density .rho. of the sample is about 1 g/cm.sup.3.
(6) The transmittance of the window of the sample container is T.sub.c
=0.63, and the efficiency of the object is .eta..sub.o =0.3. These values
are described in detail below.
If the maximum photon number n.sub.max is determined on the above-described
assumption, the following results are obtained:
(a) In a case of n.sub.max =100
##EQU11##
In this case, when the resolving power .delta. is 20 nm, the dosage exceeds
the lethal dose (.perspectiveto.1.times.10.sup.4 J/Kg).
(b) When n.sub.max is 200 or more, although it is possible to observe a
sample with lower contrast than that in the case (a), there are the
following problems;
(A) The dosage is increased and exceeds the lethal dose of cells.
(B) Since the X-rays must be further monochromatized for maintaining the
precision of photon detection, a spectral element other than the
multilayer film mirror and the like is required (described below).
(C) Since the coefficient of utilization of the X-ray source is decreased
owing to a decrease in the spectral width, a large strong X-ray source is
required.
(c) When n.sub.max is 25 or less, even if the resolving power is 20 nm,
although the dosage is reduced to a level below the lethal dose, the
detection contrast C.sub.d is 0.2 or more, the reproduced gradation number
d.sub.r is 5 or less, and the limit protein thickness is 300 nm or more.
The restrictions on the image quality and the sample detection are thus
increased, resulting in a problem in practical use.
(d) Evaluation of performance and sample treatment
A) When the thickness t.sub.p of protein is smaller than the detection
limit thickness t.sub.pd, as in (B) of the case (a), since the contrast
C.sub.d of the organism sample can be increased to a level higher than
about 0.1 by vital staining (VS) of the organism with gold having a
thickness of t.sub.gd =8.4 nm, the organism can be easily observed. The
thickness t.sub.gd of gold used for vital staining VS is determined by
substituting the linear absorption coefficient A.sub.g =24/.mu.m
[.lambda.=2.5 nm] into the equation (8) in place of A.sub.p.
B) In this way, vital staining is necessary for observation of a
low-contrast thin sample such as intracellular minute organs, virus and
the like. The X-ray exposure can be decreased by vital staining of a
specific part so as to significantly improve the contrast of a sample. For
example, although the maximum photon number n.sub.max of 100 is required
for observing a sample having contrast C of 0.1, the contrast C is
improved to 0.2 by vital staining of the sample with gold having a
thickness t.sub.gd of 8.4 nm. It is therefore found from the equation (3)
that the stained sample can be detected with the maximum number n.sub.max
of the photons detected of 25. Namely, the exposure is decreased to 1/4,
and the dosage is thus decreased to 1/4.
C) Although the dosage exceeds the lethal dose of 1.times.10.sup.4 J/Kg
when the resolving power .delta. is 20 nm, as in (C) of the case (a), in
such a case, it is effective to perform treatment such as cooling of an
organism sample for the purpose of decreasing damage to the sample and
retarding the metabolism of the cells.
(iii) Detection limit when flare is present
When a zone plate is used as an image forming element, although the first
diffracted light is used as image forming light, flare is generated by the
other orders of diffracted light. The possible influence of the flare is
the following:
Flare generally uniformly spreads over the surface of the image formed and
is thus considered as fuziness of the diaphragm or the sample, which is
caused by the optical system. The intensity of flare therefore depends
upon the intensity of irradiation, the average transmittance of the sample
and the size of visual field (the diameter of the real visual field). If
the flare coefficient is .eta..sub.f (which can be calculated, as
described below) of the optical system, the flare photon number n.sub.f
and the detected photon number n.sub.I are expressed by the following
equations:
n.sub.f .perspectiveto..eta..sub.o .multidot..eta..sub.f .multidot.T.sub.sm
.multidot.n.sub.o ( 10)
n.sub.I .perspectiveto..eta..sub.o (T.sub.s +.eta..sub.f
.multidot.T.sub.sm)n.sub.o ( 11)
From the equation (11), the number of the photons detected in each of the
pixels is expressed by the following equations:
n.sub.1 .perspectiveto..eta..sub.o (T.sub.1 +.eta..sub.f
.multidot.T.sub.sm)n.sub.o
n.sub.2 .perspectiveto..eta..sub.o (T.sub.2 +.eta..sub.f
.multidot.T.sub.sm)n.sub.o
The maximum number of the photons detected is thus expressed by the
following equation:
n.sub.max .perspectiveto..eta..sub.o (T.sub.max +.eta..sub.f
.multidot.T.sub.sm)n.sub.o
When flare is present, the contrast C.sub.I of a detectable image and the
contrast C.sub.s of a desired sample must be discriminated. The contrast
is defined by the following equations:
C.sub.I .ident..vertline.n.sub.1 -n.sub.2 .vertline./(n.sub.1 +n.sub.2)(12)
C.sub.s .ident..vertline.T.sub.1 -T.sub.2 .vertline./(T.sub.1 +T.sub.2)(13)
From the above-described relation between n.sub.1, n.sub.2 and T.sub.1,
T.sub.2, the following equation is established:
C.sub.s =C.sub.I (n.sub.1 +n.sub.2)/(n.sub.1 +n.sub.2 -2n.sub.f)(14)
As described in (i), the contrast D.sub.I of the image and the detection
limit SN ratio has the following relation:
C.sub.I.sup.2 (n.sub.1 +n.sub.2).gtoreq.(S/N).sub.d.sup.2 ( 15)
From the above equations (14) and (15) and the following equation:
n.sub.max .perspectiveto.(n.sub.1 +n.sub.2)/2,
the detection limit contrast C.sub.sd of the sample, the reproduced
gradation number d.sub.sd and the detection limit protein thickness
t.sub.pd are expressed by the following equations:
##EQU12##
Although the performance is therefore deteriorated by the presence of
flare, the detection limit can be evaluated and calculated by the above
equations.
(iv) Pulse Width Required for Dynamic Observation
If the resolving power is .delta., and the velocity is v, it is necessary
that the exposure time t.sub.x required for obtaining a clear image of a
moving object without having blurring or deformation has the following
relation:
t.sub.x .ltoreq..delta./(10.multidot.v) (19)
Since the present invention aims at an X-ray microscope having resolving
power .delta. of about 10 nm, and the maximum value V.sub.max of the
velocity of plasma streaming an the ciliary and flagellous movements is
expressed by the following equation:
V.sub.max .perspectiveto.1 mm/s,
the exposure time t.sub.x is the following:
t.sub.x .perspectiveto.1 .mu.s
Since exposure for a time of as short as 1 .mu.s cannot be easily realized
by using the scanning type of apparatus, it is rational to use a
image-formation microscopic system which uses a pulse X-ray source.
A system of exposure for a time of as short as 1 .mu.s is useful for
observing the thermal motion of the sample and the vibration of the
apparatus, and thus permits the formation of a small vibration isolator.
(v) Maximum Number of Detected Photons and Spectral Width in Photon
Counting
As described above, in order to dynamically observe a living organism, a
conventional photon counting method in time series is not used, and it is
necessary to count the photons over all the pixels for a moment of about 1
.mu.s. In order to prevent error from occurring in counting of photons,
since the energy difference caused by the wavelength difference must be
less than the energy of one photon, the following equation is established:
.DELTA.n.multidot.h.multidot..gamma.>n.sub.max
.multidot.h.multidot..DELTA..gamma.
Since the following equations are established:
.DELTA.n=1, .gamma.=c/.lambda. and
.gamma./.DELTA..gamma.=.lambda./.DELTA..lambda.,
the maximum detected photon number n.sub.max has the following relation:
n.sub.max <.lambda./.DELTA..lambda. (20)
As described above, it is preferable that the maximum number of the
detected photons is within the following range:
25.ltoreq.n.sub.max <200
The spectral width .DELTA..lambda. and the number of periods N.sub.c of the
multilayer film of the multilayer film mirror which defines the spectral
width have the following relation:
.lambda./.DELTA..lambda..perspectiveto.N.sub.c ( 21)
If 50<N.sub.c <400, therefore, the conditions for monochromatizing the
X-rays are satisfied.
If the number of periods Nc of the multilayer film is about 400 or less, it
is possible to observe a sample having a lower degree of contrast. For
example, it is possible to observe a sample having contrast which is 1/2
of that in a case of Nc of 100. However, since the dosage of the organism
is increased 4 times, the damage of the organism is increased, and the
organism frequently dies after the observation. In addition, since an
attempt can be made to further monochromatize the X-rays by increasing the
number of periods, the restrictions on the chromatic aberration of the
objective optical system are decreased, and an attempt can be thus made to
improve the performance by, for example, enlarging the effective visual
field. However, in combination of usual materials, the reflectivity is not
improved in proportion to an increase in number of the layers. In
addition, an increase in number of the layers causes a deterioration in
the efficiency of utilization of X-rays and causes the need for a stronger
X-ray source and an increase in size of the whole apparatus. The number of
periods is thus limited to about 400, and the further monochromatization
of X-rays is unsuitable for practical use from the viewpoint of a
reduction in size of the X-ray source and damage to the organism.
On the other hand, when the number of periods Nc of the multilayer film is
less than 50, it is possible to decrease the number of irradiated photons
during photon counting and decrease the damage to the organism. However,
the X-rays cannot be easily monochromatized, the chromatic aberration of
the objective optical system is made remarkable, and the counting error in
photon counting is increased. This creates a deterioration in detection
performance and a problem in practical use because only a sample having
high contrast can be observed. In addition, when the number of periods is
small, it is difficult to achieve sufficient reflectivity within the X-ray
region in the multilayer film mirror.
(vi) Sensitivity of Two-Dimensional X-Ray Imaging Element
A single photon can be detected with ideal highest sensitivity. Since the
average number of the electron hole pairs generated, which serve as
signals, is greater than the number of noise electrons in dark when the
incident X-ray photon is one within the soft X-ray region, high
sensitivity can be realized if the quantum efficiency and the vignetting
factor are close to 100%.
In a case of a solid state image sensor, if the wavelength of the X-rays
used is .lambda.=2.5 nm, and the photodetector element is Si, the average
number n.sub.p of the electron-hole pairs generated is 137, the standard
deviation .sqroot.n.sub.p F is 4, the Fano factor F is 0.12 and the number
of noise electrons in dark is 50 (electrons/pixel). The number of noise
electrons in dark can be reduced to about 10 (electron/pixel) by cooling.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing of the configuration of an image
formation-type soft X-ray microscope in accordance with the present
invention;
FIG. 2 is an explanatory view which shows a state of photon counting using
a two-dimensional X-ray imaging element;
FIG. 3 is a schematic drawing of the structure of a sample which shows the
thicknesses of water and protein;
FIG. 4 is a drawing of a basic configuration provided for explaining the
diameter of the light source of an image-forming X-ray microscope and the
efficiency of utilization thereof in the present invention;
FIG. 5 is a drawing which shows the spectral characteristics of reflected
X-rays in a rotary elliptical multilayer film mirror;
FIG. 6 is a schematic sectional view which shows the structure of the
multilayer film of a rotary elliptical multilayer film mirror;
FIG. 7 is a drawing of the optical path which shows the state of a
reflected light flux in a rotary elliptical multilayer film mirror;
FIG. 8 is a plan view of a general zone plate;
FIG. 9 is a sectional view of various zone plates;
FIG. 10 is a drawing which shows diffracted light of a zone plate;
FIG. 11 is a drawing which shows a state where flare is generated by
m-order diffracted light;
FIG. 12 is a drawing which shows a state where an image surface is curved
by a zone plate;
FIG. 13 is a plan view which shows the relation between the diagonal length
of an imaging element and the effective region of the image formed;
FIG. 14 is a schematic drawing of the structure of another embodiment using
a concave surface diffraction grating in a condenser system;
FIG. 15 is a schematic drawing of the structure of another embodiment using
a flat surface diffraction grating in a condenser system;
FIG. 16 is a schematic drawing of the structure of an embodiment which is a
combination of an optical microscope and the X-ray microscope according to
the invention;
FIG. 17 is a sectional view showing a structure of a concave reflection
mirror;
FIG. 18 is a plan view showing the structure of the concave reflecting
mirror;
FIG. 19 is a sectional view showing a structure of an objective optical
system;
FIG. 20 is a plan view showing the structure of the objective optical
system;
FIG. 21 is a schematic drawing of a configuration which shows a state where
a rotary elliptical multilayer film mirror serving as a condenser is
changed to another mirror; and
FIG. 22 is a drawing which shows the change in observation state by
exchange of an optical microscope for an X-ray microscope.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The configuration of an image formation-type X-ray microscopic apparatus in
accordance with the present invention is described below with reference to
the embodiment shown in the drawings.
In FIG. 1, the laser beams emitted from a pulse laser 1 are condensed on a
disk- or tape-shaped thin film target 5 by a condensing lens 3 through a
vacuum holding window 4 so as to cause the target 5 to generate X-rays
having necessary intensity and wavelength. The light emission of the pulse
layer 1 is controlled by a pulse control unit 2 with a desired pulse
separation (maximum, 30 Hz). The X-rays generated from the X-ray thin film
target 5 are condensed on a sample 13 in a sample container 12 by a rotary
elliptical multilayer film reflecting mirror 9. A sample image, which is
enlarged 100 times (resolving power, 100 nm) to 500 times (resolving power
20 nm), is formed on a two-dimensional X-ray imaging element 15 by using
as an image-formation optical system a phase zone plate PZP 14. For
example, it is effective to use as the two-dimensional X-ray imaging
element 15 a solid-state image sensor such as a back irradiation-type
FT-CCD.
As shown in the drawing, in this arrangement, the X-ray thin film target 5,
which is excited by the pulse laser, is disposed at the first focal point
of the rotary elliptical multilayer film reflecting mirror 9, and the
sample 13 is disposed at the second focal point thereof. The X-rays are
monochromatized by the multilayer film so that the X-rays, which are
emitted by excitation by the pulse laser, are applied to the sample, and
photon counting imaging is performed by the two-dimensional X-ray imaging
element 15.
In order to observe the sample 13, which is horizontally held, the X-rays
for irradiation and observation are arranged in the vertical direction,
and the laser beams for excitation of the X-rays are arranged in the
horizontal direction. Specifically, the angle between the target 5 and the
excitation laser beams is about 35.degree., and the angle of incident of
the X-rays on the rotary elliptical multilayer film reflecting mirror 9 is
about 65.degree.. A maintenance apparatus 6 for replacing the X-ray thin
film target 5 and removing scattered substances and the like, a diaphragm
7 and a shielding window for neutral particles and plasma are provided so
that the X-rays are applied to the sample in a predetermined direction.
The shielding window 8 also serves as a filter for adjusting the intensity
n.sub.max of the X-rays to a value of less than 200. A water cooling
apparatus is provided on the rear side of the rotary elliptical multilayer
film reflecting mirror 9 serving as a condenser for the purpose of
preventing temperature rising and deterioration, which are caused by the
absorption of the X-rays.
A field diaphragm 11 is provided just ahead of the sample container 12, the
aperture diameter thereof being changed to an appropriate value in
correspondence with the observation magnification. However, even if the
magnification is the same, the aperture diameter is changed to an
appropriate value for the purpose of preventing the occurrence of flare
and improving contrast.
The image information output from the two-dimensional X-ray imaging element
15 is processed by an image processing part 16 and then output to an image
output part 17 such as a display, a printer or the like. The image
processing part 16 has the function to adjusting the maximum number
n.sub.max of the detected photons to a value of less than 200 in linkage
with the intensity adjusting filter 8 provided on the X-ray source side.
Of the above-described components, the components from the rotary
elliptical multilayer film reflecting mirror 9 serving as a condenser to
the light-receiving surface of the two-dimensional X-ray imaging element
15 are received in a vacuum container 18 for holing them under vacuum. The
pressure in the vacuum container 18 is kept at about 10.sup.-2 Pa at which
absorption of the X-rays is negligible. Since the maintenance apparatus 6
or the like must be provided around the X-ray thin film target 5 because
scattered substances are generated around the target 5, it is necessary to
isolate the X-ray source part by another vacuum container 19.
A detailed description will now be given of the optimum specifications of
the X-ray source and the optical system for realizing photon counting
imaging for a moment of 1 pulse in a level of msec or less when the
maximum number n.sub.max of the detected photons is 100 in the
above-described arrangement.
If the efficiency of utilization of the X-ray source and the efficiency of
the optical system are determined, the final pulse intensity
(specification of the X-ray source) of the X-ray source is determined.
When a laser excitation type of X-ray source is used, the specifications
of laser are determined.
Various viewpoints are in turn described below.
(vii) Diameter and Efficiency of Utilization of Nondirectional Light Source
FIG. 4 shows the basic configuration of the image-formation X-ray
microscope. In the drawing, the X-rays emitted from the target 5 are
condensed on the sample 13 in the sample container 12 by the condenser 9,
and an enlarged image is formed on the two-dimensional X-ray imaging
element 15 through an objective optical system 14. In this embodiment, the
efficiency and the transmittance are defined as follows:
NA.sub.c : Numerical aperture on incident side of condenser
NA.sub.o : Numerical aperture on incident side of object
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