JPH0831365A - Method and device for recording and reading electron microscope image - Google Patents

Abstract

PURPOSE:To quantitatively determine the min. electron beam which does not cause deterioration of an electron microscopic image by determining the dosage of electron beams with which the signal-to-noise ratio exceeds a specified level, irradiating again the specimen with this dosage, and generating an image signal. CONSTITUTION:An electron microscopic image to represent a specimen with a low dosage of electron beams having penetrated the specimen is accumulated and recorded in a cumulative phosphor sheet, and the image signal to give the electron microscopic image is obtained by scanning the sheet photoelectrically. Then the representative value H1 of the image signals and the dosage N of electron beams cast on the specimen are determined, followed by determination of the value K1=H1/1<2/1>, and on the basis thereof the electron beam dosage N2 is determined which allows the value H2/N2 to exceed the specified value K2 when the representative value of the image signals is made H2 on the condition that the electron beams of dosage N2 based upon the previous determination are cast for irradiation. The electron beams of dosage N2 are again cast on the specimen, and its electron microscopic image is accumulated and recorded in the phosphor sheet, and the image signal to represent the electron microscopic image of the specimen is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for recording and reading an electron microscope image, and more particularly, it is possible to accumulate and record an electron microscope image on a stimulable phosphor sheet to obtain the accumulation property. The present invention relates to an electron microscope image recording / reading method and apparatus for obtaining an image signal representing an electron microscope image from a phosphor sheet.

[0002]

2. Description of the Related Art Conventionally, electron microscopes have been known in which an electron beam transmitted through a sample is refracted by an electric field or a magnetic field to obtain a magnified image of the sample. In this electron microscope, the electron beam transmitted through the sample forms a diffraction pattern of the sample on the back focal plane of the objective lens, and the diffracted waves interfere with each other again to form a magnified image of the sample. Therefore, if the magnified image is projected by the projection lens, the magnified image (scattered image) of the sample is observed, and if the back focal plane is projected, the magnified diffraction pattern of the sample is observed. If an intermediate lens is arranged between the objective lens and the projection lens, the above-mentioned magnified image (scattered image) or diffraction pattern can be optionally obtained by adjusting the focal length of this intermediate lens.

In order to observe the magnified image or diffraction pattern (hereinafter collectively referred to as a transmission electron beam image) formed as described above, conventionally, a photographic film is generally placed on the image plane of the projection lens. Exposing the transmitted electron beam image,
Alternatively, an image intensifier is arranged to amplify and project the transmission electron beam image, or a fluorescent plate is arranged to visualize the transmission electron beam image and photograph it with a television camera. However, photographic film has the drawback that it has a low sensitivity to electron beams and is troublesome to develop.On the other hand, when an image intensifier or TV camera is used, the sharpness of the image is low and the image is distorted. There is a problem that it is easy.

Further, for the transmission electron beam image as described above, image processing such as gradation processing, frequency enhancement processing, density processing, subtraction processing, addition processing, etc. for the purpose of making the image easy to see, and Fourier analysis method. Reconstruction of three-dimensional image by image, image analysis for binarization of image and particle size measurement, and processing of diffraction pattern (analysis of crystal information, lattice constant, transition, elucidation of lattice defects, etc.) In many cases, in such a case, conventionally, a microscope image obtained by developing a photographic film is read by a microphotometer and converted into an electric signal,
For example, the complicated work of performing A / D conversion of this electric signal and then processing it by a computer has been performed.

In view of the above circumstances, the applicant of the present invention is able to record and reproduce an electron microscope image with high sensitivity and high image quality, and further, in order to facilitate various kinds of processing, the electric signal carrying the microscope image is directly transmitted. A new electron microscope image recording / reproducing method to be obtained was proposed (JP-A-61-51738, JP-A-61-93539, etc.). This electron microscope image recording / reproducing method basically stores and records an electron beam transmitted through a sample in a vacuum state on a two-dimensional sensor such as a stimulable phosphor sheet that stores electron beam energy, and then records the electron beam on the two-dimensional sensor. The light is irradiated or heated to release the stored energy as light, the emitted light is photoelectrically detected to obtain an image signal, and the transmitted electron beam image of the sample is reproduced using this image signal. .

The above-mentioned two-dimensional sensor means that at least a part of the energy of the electron beam is temporarily stored when it is exposed to an electron beam, and at least a part of the stored energy is light when an external stimulus is given later. It is made of a material capable of emitting electricity, sound, etc. in a detectable form. The two-dimensional sensor is specifically described in, for example, JP-A-55-12429.
The stimulable phosphor sheet disclosed in Japanese Patent Publication No. 1994-242242 can be particularly preferably used. That is, when a certain kind of phosphor is irradiated with radiation such as an electron beam, a part of the energy of this radiation is accumulated in the phosphor, and then when the phosphor is irradiated with excitation light such as visible light, it is accumulated. The phosphor exhibits fluorescence (stimulated luminescence) depending on energy. A phosphor having such a property is called a storage phosphor. What is a stimulable phosphor sheet?
This is a sheet-shaped recording material made of the above-mentioned stimulable phosphor, and generally comprises a support and a stimulable phosphor layer laminated on this support. The stimulable phosphor layer is formed by dispersing the stimulable phosphor in an appropriate binder.When the stimulable phosphor layer is self-supporting, it becomes a stimulable phosphor sheet by itself. sell. An example of the stimulable phosphor for forming this stimulable phosphor sheet is described in detail in JP-A-61-93537.

In the above-mentioned electron microscope image recording / reproducing method, since the electron microscope image is accumulated and recorded in the two-dimensional sensor such as the stimulable phosphor sheet, it becomes possible to record the electron microscope image with high sensitivity. Therefore, the electron dose of the electron microscope can be reduced, and the damage to the sample can be reduced. Further, in this method, it becomes extremely easy to perform image processing such as gradation processing and frequency emphasis processing on the electron microscope image, and the processing of the diffraction pattern as described above,
Image analysis such as reconstruction of a three-dimensional image and binarization of an image can be performed extremely easily and quickly by inputting the electric signal to a computer.

[0008]

However, when obtaining an electron microscope image as described above, it is attempted to reduce the electron dose applied to the sample in order to protect the sample, but the electron dose is reduced. Although the degree of damage to the sample becomes smaller as it is increased, the deterioration of the obtained electron microscope image becomes larger, and the tissue or structure to be observed cannot be seen on the electron microscope image. Therefore, when taking an electron microscope image, it is necessary to preset the minimum amount of electron beam that does not deteriorate the obtained electron microscope image and perform the image taking. It depends largely on the intuition of the photographer,
It was difficult to obtain an appropriate electron dose.

In view of the above circumstances, it is an object of the present invention to provide an electron microscope image recording / reading method and apparatus capable of quantitatively obtaining the minimum electron dose without degrading the electron microscope image. .

[0010]

In the electron microscope image recording / reading method according to the present invention, an electron of a sample is irradiated by irradiating a stimulable phosphor sheet that stores electron beam energy with a predetermined amount of electron beam that has passed through the sample. Accumulate and record a microscope image in a vacuum state, then irradiate the sheet with light to release the energy of the electron beam accumulated and recorded in the sheet as light,
The emitted emission light is photoelectrically read to obtain an image signal representing an electron microscope image, and based on the obtained image signal, the representative value H _{1 of the} image signal and the electron dose N irradiated on the sample
₁ is calculated, the value k ₁ = H ₁ / √N ₁ is calculated, and the value k _{1 is} further calculated.
Based on the above, an electron whose value H ₂ / √N ₂ becomes a predetermined value k ₂ or more when the representative value of the image signal when the electron beam of the electron dose N ₂ is irradiated is H _2. Dose N ₂
Then, the sample is again irradiated with an electron beam having an electron dose of N ₂ , and the electron microscope image of the sample is stored and recorded on the stimulable phosphor sheet, and then stored and recorded on the stimulable phosphor sheet as described above. It is characterized in that an image signal representing the sample is obtained.

Here, the predetermined amount of electron beam means an electron dose N
The electron dose is sufficiently smaller than ₂ , and specifically, the electron dose N ₂ can be assumed to some extent. Therefore, an electron beam sufficiently smaller than the assumed electron dose N ₂ is used as a predetermined amount of electron beam. And it is sufficient. The representative value of the image signal is
For example, it means a value such as a difference between the maximum value and the minimum value of the obtained image signal and an average value of the image signal representing the sample.

The electron microscope image recording / reading apparatus according to the present invention is for carrying out the electron microscope image recording / reading method according to the present invention, and stores electron beam energy in the stimulable phosphor sheet as described above. Recording means for storing and recording the electron microscope image of the sample in a vacuum state by irradiating the stimulable phosphor sheet with a predetermined amount of electron beam that has passed through the sample, and the storage means for storing and recording the electron microscope image by this recording means. Means for irradiating the fluorescent phosphor sheet with light to emit the energy of the electron beam accumulated and recorded in the sheet as light, and photoelectrically reading the emitted light to obtain an image signal representing the electron microscope image. Based on the image signal obtained by this reading means, the representative value H ₁ of the image signal, the electron dose N ₁ irradiated on the sample, and the value k ₁ = H ₁ / √N ₁
Then, based on the value k ₁ , the value H when the representative value of the image signal when the electron beam with the electron dose N ₂ is irradiated is H _2.
The electron dose calculating means for calculating the electron dose N ₂ so that ₂ / √N ₂ becomes a predetermined value k ₂ or more and the electron beam of the electron dose N ₂ calculated by this electron dose calculating means are again And a control unit for controlling the recording unit so as to irradiate the sample.

[0013]

The electron microscope image recording / reading method and apparatus according to the present invention first obtains an image signal representing a sample by a sufficiently small predetermined amount of electron beam which does not destroy the sample,
Based on this image signal, the representative value H ₁ of the image signal and the electron dose N ₁ irradiated on the sample are obtained, and the value k ₁ = H ₁ / √
N ₁ or the signal-to-noise ratio is obtained, and the signal-to-noise ratio becomes a predetermined value k ₂ or more by utilizing the fact that the relationship between the electron dose and the square value of the signal-to-noise ratio changes linearly. Such an electron dose N ₂ is obtained, and the electron beam having this electron dose N ₂ is irradiated again on the sample to obtain an image signal representing this sample.

That is, it is proved as follows that the electron dose and the square value of the signal-to-noise ratio change linearly. First, the relationship between the representative value H of the image signal and the electron dose N with which the sample is irradiated can be expressed as H = aN. Here, a is a constant that varies depending on the sample, imaging conditions, and the like.
If the signal-to-noise ratio is k = H / √N, then k ² = (H / √N) ² = (a · N / √N) ² = a ² · N, and the square of the signal-to-noise ratio is , And the electron dose N and a ² change linearly with a proportional constant. Therefore, first, the sample is irradiated with a predetermined amount of electron beam to obtain the representative value H ₁ of the image signal and the electron dose N ₁ with which the sample is irradiated, and the value k
₁ = H ₁ / √N ₁ is obtained,

[0015]

[Equation 1]

Then, the proportional constant a ² is obtained from and the electron dose N ₁ . Then, from the relational expression of k ² = a ² · N, a predetermined value k ₂ is substituted into this relational expression, and the electron dose N ₂ is recognized. That is, the electron dose N ₂ is

[0017]

[Equation 2]

It is represented by

Therefore, by setting a value capable of obtaining the minimum electron dose without deteriorating the electron microscope image capable of obtaining the value k ₂ , a good image in which the structure and structure of the sample can be sufficiently observed is obtained. The electron dose to be generated can be quantitatively determined without depending on the intuition of the photographer.

By setting k ₂ to a large value, an electron microscope image with high sharpness can be obtained, and the value k ₂ can be obtained.
By setting to a small value, a smooth electron microscope image can be obtained.

[0021]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic diagram of an electron microscope image recording / reading apparatus according to an embodiment of the present invention. As shown in FIG. 1, the electron microscope image recording / reading apparatus 1 according to the embodiment of the present invention is
A stimulable phosphor sheet 10, which is arranged below the mirror body portion 1a of an ordinary electron microscope so as to belong to the same vacuum system as the image plane of the electron beam image at least during recording of the electron microscope image, and this sheet 10 Is provided with a recording / reading section 1b which is composed of excitation means for scanning with excitation light while being kept in a vacuum state and detection means for photoelectrically detecting stimulated emission light emitted from the sheet 10. A part of the mirror body portion 1a and the recording / reading portion 1b (inside the frame shown by hatching in the figure),
During operation of the electron microscope, a vacuum state is maintained by well-known means.

The mirror portion 1a includes an electron gun 3 for emitting an electron beam 2 having a uniform velocity, at least one focusing lens 4 including a magnetic lens and an electrostatic lens for narrowing the electron beam 2 on the sample surface, and the sample. The table 5 includes an objective lens 6 and a projection lens 7 similar to the focusing lens 4. The electron beam 2 transmitted through the sample 8 placed on the sample table 5 is refracted by the objective lens 6 to form an enlarged scattered image 8a of the sample 8. This enlarged scattered image 8a is image-projected by the projection lens 7 on the image plane 9 (8b in the figure). The amount of the electron beam emitted from the electron gun 3 is set sufficiently low so as not to destroy the sample.

On the other hand, the recording / reading section 1b is an endless belt-shaped stimulable phosphor sheet 10 which is wound around a cylindrical driven roller 102 as well as a cylindrical driven roller 101,
The excitation light source 11 such as a He-Ne laser tube and the excitation light beam 11a emitted from the excitation light source 11 are deflected in the width direction of the sheet 10 from the sheet 10 by the excitation means and the excitation light including the optical deflector 12 such as the galvanometer mirror. The photomultiplier 15 is provided on the exit end face of the light collector 14 that collects the emitted stimulated emission light, and includes a detection unit including a photomultiplier 15 that photoelectrically converts the stimulated emission light into an image signal representing an electron microscope image. are doing. The endless belt-shaped stimulable phosphor sheet 10 is formed by laminating a stimulable phosphor layer on the surface of a flexible endless belt-shaped support. The sheet 10 is hooked between a driving roller 101 and a driven roller 102, and a driving device (not shown)
It can be rotated in the direction of arrow A by the drive roller 101 rotated by.

In this embodiment, an endless belt-shaped stimulable phosphor sheet 10 and a driving roller 10 for moving the stimulable phosphor sheet 10.
1, driven roller 102, light collector 14 and photoelectric converter 1
Although 5 is arranged in the vacuum system, if the light condensing body 14 is arranged so that its end portion penetrates the container wall and goes out of the vacuum system, the photoelectric converter 15 can be installed outside the vacuum system. it can.

When a shutter (not shown) provided between the mirror portion 1a and the recording / reading portion 1b is opened, the stimulable phosphor sheet 1 located at the recording position (that is, the image plane 9).
Electron beam energy corresponding to the enlarged scattered image 8b of the sample is accumulated in the 0 portion. This portion of sheet 10 is then moved to the reading location by rotation of the drive roller. In this embodiment, an excitation light beam 11a for scanning the endless belt-shaped stimulable phosphor sheet 10 in the width direction is excited by a lead glass or the like by an excitation light source 11 such as a laser light source and an optical deflector 12 such as a galvanometer mirror arranged outside. The translucent wall member 19
While being incident on the sheet 10 through a, the sheet 10 is moved by the drive roller 101 in the direction (arrow A direction) perpendicular to the width direction, thereby scanning the image recording portion of the sheet 10. The stimulated emission light generated from the stimulable phosphor sheet 10 by this excitation light enters the inside of the light collector 14 from the incident end surface of the light collector 14 (the end surface facing the sheet 10), and is totally reflected therein. While being guided, it is received by the photomultiplier 15 connected to the exit end face, and the amount of stimulated emission light is photoelectrically read.

An image signal S ₁ representing an electron microscope image obtained by photoelectrically converting the stimulated emission light in the photomultiplier 15 is input to the electron dose calculating means 40. The electron dose calculation means 40 performs the following processing. First, based on the input image signal S ₁ , the sample 8
Representative value of the electron dose N ₁ and the image signals S ₁ is irradiated to the H ₁
Is required. This means that, for example, when the sample 8 is a cell having a cell membrane 8A as shown in FIG. 2, I- in FIG.
The profile of the signal value on the I line is shown in FIG. As shown in FIG. 3, the maximum value of the dose from the electron dose N ₁ applied to the sample 8 to the minimum point of the profile is used as the representative value H ₁ of the image signal S ₁ .

When the electron dose N ₁ and the representative value H _{1 of the} image signal are obtained in this way, in order to obtain the signal-to-noise ratio,

[0029]

(Equation 3)

The following calculation is performed to obtain the value k ₁ . Then, based on the value k ₁ , the optimum amount of electron dose to be applied to the sample 8 is obtained. That is, when observing a sample that is easily damaged by an electron beam with an electron microscope, the degree of damage to the sample decreases as the electron dose is reduced, but on the other hand, the deterioration of the image increases and the organization or structure desired to be observed is obtained. May not appear on the displayed image. Therefore, it is necessary to find the minimum electron dose for making the obtained electron microscope image suitable for observation. The electron dose calculating means 40 in the electron microscope image recording / reading device according to the present invention obtains this minimum electron dose.

First, as shown in FIG. 4, the horizontal axis indicates the electron dose N.
And the vertical axis is the square of the signal-to-noise ratio, that is, the value k ² , and the electron dose N ₁ and

[0032]

[Equation 4]

Plot the point P ₁ where Here, since it is known that the square value of the signal-to-noise ratio k with respect to the electron dose N changes linearly, it passes through the origin 0 and the point P
_{When a} straight line passing through ₁ is drawn, this is a graph showing the relationship between the electron dose N and the square of the signal-to-noise ratio k in this sample 8. That is, the relation between the representative value H and the electron dose N is H =
Since a · N (a is a constant that varies depending on the sample and shooting conditions),

[0034]

(Equation 5)

[0035] Therefore, the relationship between the electron dose N and the square of the signal-to-noise ratio k is linear.

Then, with the value k _2.

[0037]

(Equation 6)

The electron dose N ₂ is calculated as follows. That is,
In the graph shown in FIG.

[0039]

(Equation 7)

When the point P ₂ is obtained, the value of the electron dose at the point P ₂ becomes the electron dose N ₂ to be obtained. Here, the value k ₂ is a predetermined value for obtaining the minimum electron dose N ₂ that does not deteriorate the obtained electron microscope image, and the contrast of the obtained electron microscope image is determined by the magnitude of the value k _2. To be done.

When the electron dose N ₂ is obtained in this way, information on this electron dose N ₂ is input to the control means 41, and the control means 41 directs the electron gun 3 to emit an electron beam of the electron dose N _2. Control. As a result, an electron beam having an electron dose N ₂ is emitted from the electron gun 3 and the above-mentioned image signal S ₁
In the same manner as in the case of obtaining, the electron microscopic image showing the sample 8 is accumulated and recorded on the stimulable phosphor sheet 10, and the stimulable phosphor sheet 10 is scanned with excitation light to emit stimulated emission light, and this stimulated emission is performed. By photoelectrically converting the emitted light, an image signal S ₂ representing an electron microscope image of the sample 8 is obtained.

The image signal S ₂ is input to the image processing means 42, subjected to necessary image processing, and then reproduced by the reproducing means 43 as a visible image. This reproducing means 43 is a CR
It may be a display such as T, a recording device that performs optical scanning recording on a photosensitive film, or for that purpose, it may be one that temporarily records on a storage medium 18 such as a magnetic tape.

After the reading is completed, the image recording portion of the endless belt-shaped stimulable phosphor sheet 10 is erased zone 2.
Sent to 0. In the erasing zone 20, the erasing light emitted from the erasing light source 21 such as a fluorescent lamp provided outside the vacuum system is applied to the sheet 10 through the translucent wall member 19b. This erasing light source 21 is a stimulable phosphor sheet 1
0 is irradiated with light included in the excitation wavelength region of the phosphor, so that the afterimage accumulated in the phosphor layer of the stimulable phosphor sheet 10 or the radioactive isotope contained in the raw material of the sensor It emits elemental noise,
For example, a tungsten lamp, a halogen lamp, an infrared lamp, as shown in JP-A-56-11392,
Alternatively, a laser light source or the like can be arbitrarily selected and used.

In the above-described embodiment, the value of the difference between the minimum value and the maximum value of the profile as shown in FIG. 3 is used as the representative value of the image signal, but it is not limited to this and the image is not limited to this. Any value such as a difference value between the minimum value and the maximum value of the entire signal or a difference value between the average value and the maximum value of the signals representing the sample in the image signal may be used.

[Brief description of drawings]

FIG. 1 is a diagram showing an electron microscope image recording / reading apparatus according to an embodiment of the present invention.

FIG. 2 is a diagram showing cells as a sample.

FIG. 3 is a diagram showing a profile of an image signal along line I-I in FIG.

FIG. 4 is a graph showing the relationship between electron dose N and value k ^2.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Electron microscope 2 Electron beam 3 Electron gun 9 Image plane of electron microscope 10 Storage phosphor sheet 11 Excitation light source 11a Excitation light beam 12 Optical deflector 14 Condenser 15 Photomultiplier 40 Electron dose calculation means 41 Control means 42 Image processing means 43 Reproducing means S ₁ , S ₂ image signal N electron dose

Claims (2)

[Claims] 1. An electron microscopic image of a sample is accumulated and recorded in a vacuum state by irradiating a stimulable phosphor sheet that accumulates electron beam energy with a predetermined amount of an electron beam that has passed through the sample. Image signal representing the electron microscope image by irradiating the fluorescent phosphor sheet with light to cause the energy of the electron beam accumulated and recorded in the sheet to be emitted as light and photoelectrically reading the emitted light emitted. Then, based on the image signal, a representative value H _{1 of the} image signal and an electron dose N ₁ irradiated to the sample are obtained, a value k ₁ = H ₁ / √N ₁ is obtained, and the value k ₁ is obtained. Based on this, the value H ₂ / √ when the representative value of the image signal when the electron beam with the electron dose N ₂ is irradiated is H _2.
The calculated electron beam dose N ₂ as N ₂ becomes the predetermined value k ₂ than the predetermined stimulable phosphor electron microscope image of the sample by irradiating the electron beam of the electron dose N ₂ again sample Accumulation and recording on a sheet, then, the stimulable phosphor sheet is irradiated with light to cause the electron beam energy of the electron dose N ₂ accumulated and recorded on the sheet to be emitted as light, and the emitted emission An electron microscope image recording / reading method comprising photoelectrically reading light to obtain an image signal representing the electron microscope image. 2. A recording means for accumulating and recording an electron microscope image of the sample in a vacuum state by irradiating a stimulable phosphor sheet for accumulating electron beam energy with a predetermined amount of an electron beam transmitted through the sample, By irradiating the stimulable phosphor sheet on which the electron microscope image is accumulated and recorded by the recording means, the energy of the electron beam accumulated and recorded on the sheet is emitted as light.
Reading means for photoelectrically reading the emitted emitted light to obtain an image signal representing the electron microscope image, and a representative value H _{1 of the} image signal and the sample based on the image signal obtained by the reading means. The electron dose N ₁ and the value k ₁ = H ₁ / √N ₁ are calculated, and the representative value of the image signal when the electron beam of the electron dose N ₂ is irradiated is H ₂ based on the value k _1. And an electron dose N ₂ calculated so that the value H ₂ / √N ₂ becomes equal to or more than a predetermined value k ₂ determined in advance, and an electron dose N calculated by the electron dose calculator. _2. An electron microscope image recording / reading apparatus comprising: a control unit that controls the recording unit to irradiate the sample with the _second electron beam.