- Email: [email protected]
Application of the “imaging plate” to TEM image recording
Ultramicroscopy 25 (1988) 195-202 North-Holland, Amsterdam
195
APPLICATION OF THE "IMAGING PLATE" TO TEM IMAGE RECORDING N. MORI Fuji Photo Film Co., Ltd., Miyanodai Kaisei-machi, Ashigarakami-gun, Kanagawa 258, Japan
T. OIKAWA JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196, Japan
T. KATOH and J. MIYAHARA Fuji Photo Film Co., Ltd., Miyanodai Kaisei-machi, Ashigarakami-gun, Kanagawa 258. Japan
and Y. HARADA JEOL Ltd., 1-2 Musashino 3-chome, Akishima, TokTo 196, Japan Received 8 January 1988
The "imaging plate" is a highly sensitive image recording plate for X-ray radiography, which is coated x~ith photo-stimulable phosphor. The imaging plate is exposed to electrons in a transmission electron rmcroscope. Its fundamental properties (sensitivity, dynamic range and sharpness) have been estimated in detail. Also. the image quality of the imaging plate for some specimens in a transmission electron microscope has been estimated. As a result, it has been ascertained that the imaging plate has superior properties and high practicability as an image recording material in a transmission electron microscope.
1. Introduction
Photo film coated with photographic emulsion has been used mainly as an image recording material in a transmission electron microscope (TEM) for over half a century since the TEM was invented. Lately, techniques to reduce electron irradiation damage to a specimen and highly sensitive image detectors to observe an image with a lower electron dose are required, because there is an increasing need for observation of electron-sensitive specimens such as organic crystals and biological specimens (DNA, etc.). A specimen-cooling technique (cryo-protection) [1,2] and the minimum dose system (MDS) [3,4]. which is a technique to irradiate the field of view with an electron beam only while taking a photograph~ are
used to reduce electron irradiation damage. The TV camera [1] is an image detector differem front photo film, and it has high sensitivity. However, its image quality is not so high. The "imaging plate" (IP) was developed as an image recording material in X-ray radiography (computed radiography (CR) system [5]). It has higher sensitivity than conventional X-ray film and higher image quality than the other electrical detector. Since the IP produces image data as electrical signals, it is convenient to process and file the images. The IP was applied to TEM image recording in a preliminary manner [6-81. In this paper, the fundamental properties of the IP for electrons and the image quality of the IP in TEM for some ~pecimens are reported. Also, some of its ad-
0304-3991/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
196
N, Mori et aL / Application of " imaging plate" to TEM image recording
¢ PRINTIN(I
vantages (high sensitivity, ~ide dynamic range, variable image contrast, easy-to-digitize image signals and ease of handling) have been ascertained.
2. Outline of IP imaging system 2.1. Principle of image recording on the IP
t
m -, 1 IMA(IIN~PLATE(IP} EXPOSURE
In this section the IP is introduced briefly, since detailed papers [9,10] are already published. Fig. 1 shows the schematic diagram of the principle of image recording on the IP. The IP is a flexible plate with a thickness from 0.3 to 0.5 mm, which is composed of a protective layer, phosphor layer and support. The main part of the IP is the phosphor layer, which is made of photo-stimulatable phosphor. When the IP is irradiated with radioactive rays (including X-rays and electrons), the energy of the rays is stored in the phosphor. And then, by irradiating the IP with light (He-Ne laser light is used in the CR system), the energy is emitted in the form of light. This phenomenon is called "'photo-stimulated luminescence". In the CR system, BaFX : Eu (X = C1, Br, I) is used as phosphor. The material emits a light which has the maximum intensity at wavelength X = 390 nm. The phosphor, which has a 5 gm grain size, is applied to the plastic base plate, together with an organic binder. The IP is covered with a trans/
PROTECT]'JELAYER
d ~-z~ ~2-~~-;-~ ~'~,~~ ,, ~ ,, '~,,F- PHOTOSTIMULABLEPHOSPHDR "~/~P/////,P//~//~//~/r'- SUPPORT
it
.oH READING
ERASINea
l
Fig. 2. Schematic diagram of the total system of the IP including the TEM. The IP is reusable in the sequential process of exposure in the TEM ~ reading in the reader --* erasing.
parent protective layer in order to protect the phosphor layer.
2.2. Image-reading system A reader, processor, printer and eraser are needed to utilize the image recorded on the IP. Fig. 2 shows the schematic diagram of the total system including the TEM. In the reader, H e - N e laser light is scanned over the IP, the emitted blue light is then collected efficiently and guided to a photomultiplier tube (PMT) by a light pipe. In the PMT the light is converted into an electrical signal and processed (e.g. gray-level processing, spatial frequency processing, etc.) in the processor. The final image is printed in the printer. The image energy remaining in the IP is erased by irradiating it with enough light in the eraser, thus rendering the IP reusable.
ELECTRONS I
1 | I |
~1
I
1.1_
LASER BEArl SCANNING ,
'
'
'~"~,l# ~ ' ~
LUI 11 IIt'.~ Lr'~"ILB
3. Experiment
0
LIGHT =" t t l l / / t i i / i t / l J
ERASING
Fig. 1. Schematic diagram 9f processes of recording, reading, and erasing an image on the IP.
3.1. IP Two types of IPs were used in this work. One is IP-A which has a 150 ~tm thick phosphor layer, and the other is IP-B, which has a 50 um thick phosphor layer. Since the size (82 mm x ,18 mm) of both IPs has been made the same as that of photo film for TEM, the IPs can be exposed in the camera chamber of TEM without any modification of that camera chamber.
N. Mort et al. / App#catien of "'imaging plate " to T E M image recording
Table 1 Properties of the IPs used in this experiment
197
PL-200WP, and it is automatically developed in the developer.
Properties
IP-A
IP-B
Thickness of protective layer (ttm) Thickness of phosphor layer (ix m) Thickness of support (~tm) Relative sensitivity for electron, compared with IP-A at 100 kV at 200 kV Reading pixel size (/tin) Sharpness at response 0.5 (line pairs/ram)
10 150 250
50 250
4. Results and discussion
4.1. Fundamental properties 1 1 100 x 1130 3.0
0.5 0.3 25 x 25 5.0
The evacuation time of the camera chamber with the IP was about half that with photo film, because the outgas from the IP was less than that from the film. Table 1 shows the fundamental properties of the IPs used in this experiment.
3.2. T E M A JEM-12OOEX (120 kV TEM), a 2000FX (200 kV TEM) and a 4000FX (400 kV TEM) were used.
3.3. Reader and printer The FCR-101, a reader and printer designed for X-ray radiography, was used for the IP-A. In the reader, the pixel size is 100 # m x 100 #m, gray level in 8 bits (256 stages), and reading time is about 90 s for the size of X-ray radiography (201 m m x 254 ram). In the printer, the pixel size is 100 / , m x 100 /,m and gray level is 10 bits (1024 stages). The printing material is transparent film. In this experiment, the final image was printed on photographic printing paper (Fuji Bromide paper) from the film, by the conventional photographic treatment. For the I£-B a prototype drum-scanner-type reader and printer was used. Its pixel size is 25 p.m × 25/tin, gray level is 12 bits (4096 stages) and reading time is 1080 s. It is not difficult to improve the reading time to the same level as the FCR-101. The pixel size and gray level of the printer are the same as those of the reader. The printing material is photographic p f m i n g paper
4.1.1. Sensitivity and dynamic range Fig. 3 shows the signal intensity from the IP-A as a function of the electron dose on the IP at 200 kV. The curve for the film shows the optical density of the photo film (Fuji FG film). The IP has a sensitivity about three orders higher than Fuji FG film, good linearity in electron dose and a wide dynamic range of about four orders. The superior properties of the IP are due to the properties of the phosphor and the PMT. The IP has a high stopping power for electrons because its phosphor layer is thick (150/am), and has a high density. Also, the IP has a high efficiency of electron-photon conversion. The energy ratio of accelerated electrons (about 100 keV) in TEM to photons (several eV) is about four orders. Therefore, about 100 photons are generated by a single electron on the assumption that electron-photon conversion efficiency is 1% [11]. It is sufficient signal intensity to produce an image with a good signal-to-noise ratio iS/N). The IP is convenient for quantitative measurement of the electron intensity because of its superior properties, which makes chemical processing unnecessary.
10 5 104
200 kV
f/l"
>..
10 3
IP4L "
la_
4___ 7©
Jlt
>-
7 r.
,g © 10-15 10-14 10 -~'~ 10-~2 10-:: 10 -!c 10-9 ELECTRON DOSE ,C,,crnZ
Fig. 3. Signal intensity from the IP-A and optical density of the Fuji FG filnl as a function of electron dose at 200 kV.
198
N. Mori et aL / Appfication of"imaging plate" to TEM image recording
4.1.2. Dependence of sensitivity on accelerating voltage The dependence of the sensitivity on accelerating voltage was measured for IP-A and photo film. The sensitivity of the IP is defined by the photon intensity for the IP. The sensitivity of the photo film (Slam) is defined as the reciprocal electron dose which renders an optical density 1.0. The sensitivity of the photo film decreases with increasing accelerating voltage. The reason is that the electrons passing through the photo film increase with increasing accelerating voltage. The sensitivity of the IP-A does not depend on the accelerating voltage in the range of 100-400 kV measured. The reason is considered to be the following: The phosphor layer of IP-A is thick. The penetration range for 400 kV is estimated at 230/xm by using the Katz-Penfold equation [12] and using the phosphor density of p = 5.14. This implies that 88% of the incident electrons are caught in the phosphor layer. For this thickness of the phosphor layer, the electrons caught in the layer increase with accelerating voltage. However, photons generated in the deeper layer in which electrons are caught are absorbed in the phosphor layer. Therefore, the aFparent signal intensity is not dependent on the accelerating voltage. This is an advantage for a high-voltage electron microscope (HVEM). 4.1.3. Sharpness The sharpness of both IP-A and IP-B was estimated by measuring the response of a pattern made by a 30 ~m thick nickel grid, as shown in fig. 4a. The experiment was carried out at 120 kV, and the X-rays generated on the nickel grid were ascertained to be negligible. The edge effect [13] did not occur in this experiment. Fig. 4b shows an ideal pattern which should be exposed on the IP. Fig. 4c shows the response recorded on tke IP. The lowering of the amplitude of response in the region of higher spatial frequency shows deterioration of the sharpness. Therefore, the sharpness is estimated by the response R , - that is, the ratio of the amplitude at spatial frequency i to that at zero frequency. Fig. 4d shows the measured response as a function of
(a) ELEOTROXBS/~ ~, ~, $ $ $ ~, METALMASK
. . . . . . . . . . . . .
(b)
ELECTRONBEAM PATTERN _nJ~J-L!~J]~
(C}
P~E,SPO~.SOF F_.IP Ai R i -" - Ao
'
to ~
(d)
SAMPLING ZP' ®
25jura
IP-B
0,5 ¢,0 uJ r'r"
0
-
o
m
2.0
__,
I
m
i
4.0
s.o
8,0
SPATIAL FP,EQUENCY(Ip/mrn) Fig. 4. Measured response of IP-A and IP-B as a function of
spatial frequencyat 120 kV. (a) Mask pattern made of 30 ~m thick nickel.(b) Ideal pattern whichshould be exposed on the IP. (c) Response recorded on the IP. (d) Measured response c~,'-veas a function of the spatial frequency.
the spatial frequency. The sharpness of IP-B is higher than that of IP-A. IP-B has a thinner phosphor layer (50 Fro) than IP-A, and is read by a smaller pixel size (25 #m) than IP-A. The sharpness of IP can be improved by making the phos. phor layer thinner and setting the reading pixel smaller; however, the sensitivity becomes lowcr. Therefore, there is room for the IP and reader optimally designed for the TEM.
4.2. Appfication data of practical specimens Fig. 5 shows 1.3 nm lattice images of chlorinated copper ~h,hol . . . . . ";"'~ recorded _ the IP-A. The images were taken under the condition of 200 kV accelerating voltage. Fig. 5a was taken at × 300,000 direct magnification, 2 × 10- ~3 C / c m 2 electron dose on the IP and 1200 electrons/nm 2 on the specimen. Fig. 5b was taken at ,'<600,000 direct magnification, 1.3 × 10-14 C / c m 2 electron dose on the IP and 300 electrons/nm 2 on the specimen. The lattices are visible faintly as
N. Mori er al. / Applica:zon of "imaging plate" to TEM image recording
%6
199
.~"
Fig. 5. 1.3 nm lattice images of chlorinated copper phthalocyanine recorded on the IP-A. The images were taken with JEM-2000FX under the conditions of 200 kV accelerating voltage. (a) Taken at x 300,000 direct magnification. 2 x 10-~3 C / c m 2 electron dose on the IP and 1200 electrons/rim 2 on the specimen. (b) Taken at x 600,000 direct magnification. 1.3 x 1 0 - ~4 C / c m 2 electron dose on the IP and 300 electrons/rim 2 on the specimen.
indicated by arrows, because the images were blurred by the quantum noise due to low electron dose. With photo film, a 2700 electrons/nm 2 electron dose on the specimen is the lower limitation, because of its lower sensitivity. Fig. 6 shows convergent beam electron diffraction (CBED) patterns of Si(lll), taken on the IP-B and the FG film. The FG film was develcped in a low-contrast developer. Since the sensitivity
of the IP is different from that of the FG film, the patterns were taken with optimum dose for each material. The conditions for exposure, reading the IP, and developing the film are mentioned in the figure caption. The whole pattern is well recorded on the IP (fig. 6a) because of its wide dynamic range, while only the center of the pattern is recorded on the FG film (fig. 6b) due to its narrow dynamic range.
fig, 6. A comparison of C B E D patterns of Si(111) recorded on the IP-B and F G film at 100 kV (with JEM-20OOFX). (a) Recorded on the IP-B, The pattern was recorded at a mean electron dose of 8 x 10 -12 C / c m 2 on the IP, and the dynamic range in reading is 3.6 orders. (b) Recorded on the F G film. The pattern was recorded at a mean electron dose of I x 10- m C/cm: on the film. The film was developed for 4 rain at 20 o C in a developer obtained by diluting Konidor Soft with water at l : 2.
200
N. Mori et al. / Application of "imaging plate" to TEM image recording
Fig. 7. Neurohypophysis of a rat recorded on the IP-B. The specimen was stained with uranyl acetate and lead citrate. "[he image was taken with JEM-1200EX at an accelerating voltage of 120 kV, direct magnification of x 3,000 and electron dose of 8 x 10-13 C/cm 2 on the IP. (a) Original image taken on the IP. (b) Image with spatial frequency (2.7 mm- 1) erahancement.
Fig. 7 shows the neurohypophysis of a rat as a biological example. Fig. 7a shows the original image taken on the IP-B. Fig. 7b shows an image spatial frequency in the vicinity of 2.7 mm-~ on the IP enhanced by image processor. The apparent image quality is good enoagh for practical use in the biological field.
(4) Digitized image, which is convenient for image processing, filing and retrieval. (5) Easy handling: (a) no ,reed of dark room; (b) automatic imaging. The next target is to optimally design the IP for TEM.
Acknowledgements 5. Conclusions The IP, which was developed as an image recording material for X-ray radiography, has been applied to TEM image recording. As a result, the superior properties of the IP for electrons have been ascertained. Also, its high practicability as an image recording material in TEM has been ascertained using some specimens. The IP has some advantages in its treatment and handling, in addition to its f,mdamental properties. The advantages of IP as an image recording material in TEM are listed as follows: (1) High sensitivity. (2) Wide dynamic range. (3) Variable contrast.
The authors are grateful to Dr. Y. Fujiyoshi of Protein Engineering Research Institute for his useful discussion, and to Mr. M. Hakamata of Fuji Photo Film Co., Ltd., for helping operate the reader and printer for the IP-B.
References [1] L. Reimer, Transmission Electron Microscopy (Springer, Berlin, 1984). [2] E. Zeitler, Ultramicro'vopy 10 (1982) 1. [3] Y. Fujiyoshi, T. Kobayasti, N. Uyeda, Y. Ishida and Y. Harada, Ultramicroscopy 5 (1980) 459. [4] G. Wrigley, E. Brown and P.K. Chillingworth, J. Microscopy 130 (1983) 225.
N. Mori et at / Application of "'imaging plate'" to TF,M image recording [5] M. Sonoda et ai., Radiology 148 (1983) 833, {61 S. lchihara et al., J. Electron Microsc. 33 (1984) 255. [7] N. Mori et al., in: Proc. l l t h Intern. Conf. on Electron Microscopy, Kyoto, 1986, Eds. T. lmura, S. Maruse and T. Suzuki (Japan. Soc. Electron Microscopy, Tokyo, 1986) Vol. I, p. 29. [8] T. Oikawa et al., in: Proc. l l t h Intern. Conf. on Electron Microscopy, Kyoto, 1986, Eds. T. Imura, S, Maruse and T. Suzuki (Japan. Soc. Electron Microscopy, Tokyo, 1986) Vol. 1, p. 439.
201
[9] K. Takahashi, K. Kohda, J. Miyahara, Y. Kanemitsu, K. Amitani and S. Shionoya. J. Luminescence 31 & 32 (/984) 266. [10] K. Takahashi, J. Miyahara and Y. Shibahara, J. Electrochem. Soe. 132 (1995) 1492. [111 D.J. Perry and J.G. Holt, Med. Phys. 7 (1980) 207. [12] L. Katz and A.S. Penfold, Rev. Mod. Phys. 24 (1952) 30. [131 G.W. Ludwig and J.D. Kingsley, J. Electrochem. SOc. Solid Slate Sci. 117 (1970) 348.
195
APPLICATION OF THE "IMAGING PLATE" TO TEM IMAGE RECORDING N. MORI Fuji Photo Film Co., Ltd., Miyanodai Kaisei-machi, Ashigarakami-gun, Kanagawa 258, Japan
T. OIKAWA JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196, Japan
T. KATOH and J. MIYAHARA Fuji Photo Film Co., Ltd., Miyanodai Kaisei-machi, Ashigarakami-gun, Kanagawa 258. Japan
and Y. HARADA JEOL Ltd., 1-2 Musashino 3-chome, Akishima, TokTo 196, Japan Received 8 January 1988
The "imaging plate" is a highly sensitive image recording plate for X-ray radiography, which is coated x~ith photo-stimulable phosphor. The imaging plate is exposed to electrons in a transmission electron rmcroscope. Its fundamental properties (sensitivity, dynamic range and sharpness) have been estimated in detail. Also. the image quality of the imaging plate for some specimens in a transmission electron microscope has been estimated. As a result, it has been ascertained that the imaging plate has superior properties and high practicability as an image recording material in a transmission electron microscope.
1. Introduction
Photo film coated with photographic emulsion has been used mainly as an image recording material in a transmission electron microscope (TEM) for over half a century since the TEM was invented. Lately, techniques to reduce electron irradiation damage to a specimen and highly sensitive image detectors to observe an image with a lower electron dose are required, because there is an increasing need for observation of electron-sensitive specimens such as organic crystals and biological specimens (DNA, etc.). A specimen-cooling technique (cryo-protection) [1,2] and the minimum dose system (MDS) [3,4]. which is a technique to irradiate the field of view with an electron beam only while taking a photograph~ are
used to reduce electron irradiation damage. The TV camera [1] is an image detector differem front photo film, and it has high sensitivity. However, its image quality is not so high. The "imaging plate" (IP) was developed as an image recording material in X-ray radiography (computed radiography (CR) system [5]). It has higher sensitivity than conventional X-ray film and higher image quality than the other electrical detector. Since the IP produces image data as electrical signals, it is convenient to process and file the images. The IP was applied to TEM image recording in a preliminary manner [6-81. In this paper, the fundamental properties of the IP for electrons and the image quality of the IP in TEM for some ~pecimens are reported. Also, some of its ad-
0304-3991/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
196
N, Mori et aL / Application of " imaging plate" to TEM image recording
¢ PRINTIN(I
vantages (high sensitivity, ~ide dynamic range, variable image contrast, easy-to-digitize image signals and ease of handling) have been ascertained.
2. Outline of IP imaging system 2.1. Principle of image recording on the IP
t
m -, 1 IMA(IIN~PLATE(IP} EXPOSURE
In this section the IP is introduced briefly, since detailed papers [9,10] are already published. Fig. 1 shows the schematic diagram of the principle of image recording on the IP. The IP is a flexible plate with a thickness from 0.3 to 0.5 mm, which is composed of a protective layer, phosphor layer and support. The main part of the IP is the phosphor layer, which is made of photo-stimulatable phosphor. When the IP is irradiated with radioactive rays (including X-rays and electrons), the energy of the rays is stored in the phosphor. And then, by irradiating the IP with light (He-Ne laser light is used in the CR system), the energy is emitted in the form of light. This phenomenon is called "'photo-stimulated luminescence". In the CR system, BaFX : Eu (X = C1, Br, I) is used as phosphor. The material emits a light which has the maximum intensity at wavelength X = 390 nm. The phosphor, which has a 5 gm grain size, is applied to the plastic base plate, together with an organic binder. The IP is covered with a trans/
PROTECT]'JELAYER
d ~-z~ ~2-~~-;-~ ~'~,~~ ,, ~ ,, '~,,F- PHOTOSTIMULABLEPHOSPHDR "~/~P/////,P//~//~//~/r'- SUPPORT
it
.oH READING
ERASINea
l
Fig. 2. Schematic diagram of the total system of the IP including the TEM. The IP is reusable in the sequential process of exposure in the TEM ~ reading in the reader --* erasing.
parent protective layer in order to protect the phosphor layer.
2.2. Image-reading system A reader, processor, printer and eraser are needed to utilize the image recorded on the IP. Fig. 2 shows the schematic diagram of the total system including the TEM. In the reader, H e - N e laser light is scanned over the IP, the emitted blue light is then collected efficiently and guided to a photomultiplier tube (PMT) by a light pipe. In the PMT the light is converted into an electrical signal and processed (e.g. gray-level processing, spatial frequency processing, etc.) in the processor. The final image is printed in the printer. The image energy remaining in the IP is erased by irradiating it with enough light in the eraser, thus rendering the IP reusable.
ELECTRONS I
1 | I |
~1
I
1.1_
LASER BEArl SCANNING ,
'
'
'~"~,l# ~ ' ~
LUI 11 IIt'.~ Lr'~"ILB
3. Experiment
0
LIGHT =" t t l l / / t i i / i t / l J
ERASING
Fig. 1. Schematic diagram 9f processes of recording, reading, and erasing an image on the IP.
3.1. IP Two types of IPs were used in this work. One is IP-A which has a 150 ~tm thick phosphor layer, and the other is IP-B, which has a 50 um thick phosphor layer. Since the size (82 mm x ,18 mm) of both IPs has been made the same as that of photo film for TEM, the IPs can be exposed in the camera chamber of TEM without any modification of that camera chamber.
N. Mort et al. / App#catien of "'imaging plate " to T E M image recording
Table 1 Properties of the IPs used in this experiment
197
PL-200WP, and it is automatically developed in the developer.
Properties
IP-A
IP-B
Thickness of protective layer (ttm) Thickness of phosphor layer (ix m) Thickness of support (~tm) Relative sensitivity for electron, compared with IP-A at 100 kV at 200 kV Reading pixel size (/tin) Sharpness at response 0.5 (line pairs/ram)
10 150 250
50 250
4. Results and discussion
4.1. Fundamental properties 1 1 100 x 1130 3.0
0.5 0.3 25 x 25 5.0
The evacuation time of the camera chamber with the IP was about half that with photo film, because the outgas from the IP was less than that from the film. Table 1 shows the fundamental properties of the IPs used in this experiment.
3.2. T E M A JEM-12OOEX (120 kV TEM), a 2000FX (200 kV TEM) and a 4000FX (400 kV TEM) were used.
3.3. Reader and printer The FCR-101, a reader and printer designed for X-ray radiography, was used for the IP-A. In the reader, the pixel size is 100 # m x 100 #m, gray level in 8 bits (256 stages), and reading time is about 90 s for the size of X-ray radiography (201 m m x 254 ram). In the printer, the pixel size is 100 / , m x 100 /,m and gray level is 10 bits (1024 stages). The printing material is transparent film. In this experiment, the final image was printed on photographic printing paper (Fuji Bromide paper) from the film, by the conventional photographic treatment. For the I£-B a prototype drum-scanner-type reader and printer was used. Its pixel size is 25 p.m × 25/tin, gray level is 12 bits (4096 stages) and reading time is 1080 s. It is not difficult to improve the reading time to the same level as the FCR-101. The pixel size and gray level of the printer are the same as those of the reader. The printing material is photographic p f m i n g paper
4.1.1. Sensitivity and dynamic range Fig. 3 shows the signal intensity from the IP-A as a function of the electron dose on the IP at 200 kV. The curve for the film shows the optical density of the photo film (Fuji FG film). The IP has a sensitivity about three orders higher than Fuji FG film, good linearity in electron dose and a wide dynamic range of about four orders. The superior properties of the IP are due to the properties of the phosphor and the PMT. The IP has a high stopping power for electrons because its phosphor layer is thick (150/am), and has a high density. Also, the IP has a high efficiency of electron-photon conversion. The energy ratio of accelerated electrons (about 100 keV) in TEM to photons (several eV) is about four orders. Therefore, about 100 photons are generated by a single electron on the assumption that electron-photon conversion efficiency is 1% [11]. It is sufficient signal intensity to produce an image with a good signal-to-noise ratio iS/N). The IP is convenient for quantitative measurement of the electron intensity because of its superior properties, which makes chemical processing unnecessary.
10 5 104
200 kV
f/l"
>..
10 3
IP4L "
la_
4___ 7©
Jlt
>-
7 r.
,g © 10-15 10-14 10 -~'~ 10-~2 10-:: 10 -!c 10-9 ELECTRON DOSE ,C,,crnZ
Fig. 3. Signal intensity from the IP-A and optical density of the Fuji FG filnl as a function of electron dose at 200 kV.
198
N. Mori et aL / Appfication of"imaging plate" to TEM image recording
4.1.2. Dependence of sensitivity on accelerating voltage The dependence of the sensitivity on accelerating voltage was measured for IP-A and photo film. The sensitivity of the IP is defined by the photon intensity for the IP. The sensitivity of the photo film (Slam) is defined as the reciprocal electron dose which renders an optical density 1.0. The sensitivity of the photo film decreases with increasing accelerating voltage. The reason is that the electrons passing through the photo film increase with increasing accelerating voltage. The sensitivity of the IP-A does not depend on the accelerating voltage in the range of 100-400 kV measured. The reason is considered to be the following: The phosphor layer of IP-A is thick. The penetration range for 400 kV is estimated at 230/xm by using the Katz-Penfold equation [12] and using the phosphor density of p = 5.14. This implies that 88% of the incident electrons are caught in the phosphor layer. For this thickness of the phosphor layer, the electrons caught in the layer increase with accelerating voltage. However, photons generated in the deeper layer in which electrons are caught are absorbed in the phosphor layer. Therefore, the aFparent signal intensity is not dependent on the accelerating voltage. This is an advantage for a high-voltage electron microscope (HVEM). 4.1.3. Sharpness The sharpness of both IP-A and IP-B was estimated by measuring the response of a pattern made by a 30 ~m thick nickel grid, as shown in fig. 4a. The experiment was carried out at 120 kV, and the X-rays generated on the nickel grid were ascertained to be negligible. The edge effect [13] did not occur in this experiment. Fig. 4b shows an ideal pattern which should be exposed on the IP. Fig. 4c shows the response recorded on tke IP. The lowering of the amplitude of response in the region of higher spatial frequency shows deterioration of the sharpness. Therefore, the sharpness is estimated by the response R , - that is, the ratio of the amplitude at spatial frequency i to that at zero frequency. Fig. 4d shows the measured response as a function of
(a) ELEOTROXBS/~ ~, ~, $ $ $ ~, METALMASK
. . . . . . . . . . . . .
(b)
ELECTRONBEAM PATTERN _nJ~J-L!~J]~
(C}
P~E,SPO~.SOF F_.IP Ai R i -" - Ao
'
to ~
(d)
SAMPLING ZP' ®
25jura
IP-B
0,5 ¢,0 uJ r'r"
0
-
o
m
2.0
__,
I
m
i
4.0
s.o
8,0
SPATIAL FP,EQUENCY(Ip/mrn) Fig. 4. Measured response of IP-A and IP-B as a function of
spatial frequencyat 120 kV. (a) Mask pattern made of 30 ~m thick nickel.(b) Ideal pattern whichshould be exposed on the IP. (c) Response recorded on the IP. (d) Measured response c~,'-veas a function of the spatial frequency.
the spatial frequency. The sharpness of IP-B is higher than that of IP-A. IP-B has a thinner phosphor layer (50 Fro) than IP-A, and is read by a smaller pixel size (25 #m) than IP-A. The sharpness of IP can be improved by making the phos. phor layer thinner and setting the reading pixel smaller; however, the sensitivity becomes lowcr. Therefore, there is room for the IP and reader optimally designed for the TEM.
4.2. Appfication data of practical specimens Fig. 5 shows 1.3 nm lattice images of chlorinated copper ~h,hol . . . . . ";"'~ recorded _ the IP-A. The images were taken under the condition of 200 kV accelerating voltage. Fig. 5a was taken at × 300,000 direct magnification, 2 × 10- ~3 C / c m 2 electron dose on the IP and 1200 electrons/nm 2 on the specimen. Fig. 5b was taken at ,'<600,000 direct magnification, 1.3 × 10-14 C / c m 2 electron dose on the IP and 300 electrons/nm 2 on the specimen. The lattices are visible faintly as
N. Mori er al. / Applica:zon of "imaging plate" to TEM image recording
%6
199
.~"
Fig. 5. 1.3 nm lattice images of chlorinated copper phthalocyanine recorded on the IP-A. The images were taken with JEM-2000FX under the conditions of 200 kV accelerating voltage. (a) Taken at x 300,000 direct magnification. 2 x 10-~3 C / c m 2 electron dose on the IP and 1200 electrons/rim 2 on the specimen. (b) Taken at x 600,000 direct magnification. 1.3 x 1 0 - ~4 C / c m 2 electron dose on the IP and 300 electrons/rim 2 on the specimen.
indicated by arrows, because the images were blurred by the quantum noise due to low electron dose. With photo film, a 2700 electrons/nm 2 electron dose on the specimen is the lower limitation, because of its lower sensitivity. Fig. 6 shows convergent beam electron diffraction (CBED) patterns of Si(lll), taken on the IP-B and the FG film. The FG film was develcped in a low-contrast developer. Since the sensitivity
of the IP is different from that of the FG film, the patterns were taken with optimum dose for each material. The conditions for exposure, reading the IP, and developing the film are mentioned in the figure caption. The whole pattern is well recorded on the IP (fig. 6a) because of its wide dynamic range, while only the center of the pattern is recorded on the FG film (fig. 6b) due to its narrow dynamic range.
fig, 6. A comparison of C B E D patterns of Si(111) recorded on the IP-B and F G film at 100 kV (with JEM-20OOFX). (a) Recorded on the IP-B, The pattern was recorded at a mean electron dose of 8 x 10 -12 C / c m 2 on the IP, and the dynamic range in reading is 3.6 orders. (b) Recorded on the F G film. The pattern was recorded at a mean electron dose of I x 10- m C/cm: on the film. The film was developed for 4 rain at 20 o C in a developer obtained by diluting Konidor Soft with water at l : 2.
200
N. Mori et al. / Application of "imaging plate" to TEM image recording
Fig. 7. Neurohypophysis of a rat recorded on the IP-B. The specimen was stained with uranyl acetate and lead citrate. "[he image was taken with JEM-1200EX at an accelerating voltage of 120 kV, direct magnification of x 3,000 and electron dose of 8 x 10-13 C/cm 2 on the IP. (a) Original image taken on the IP. (b) Image with spatial frequency (2.7 mm- 1) erahancement.
Fig. 7 shows the neurohypophysis of a rat as a biological example. Fig. 7a shows the original image taken on the IP-B. Fig. 7b shows an image spatial frequency in the vicinity of 2.7 mm-~ on the IP enhanced by image processor. The apparent image quality is good enoagh for practical use in the biological field.
(4) Digitized image, which is convenient for image processing, filing and retrieval. (5) Easy handling: (a) no ,reed of dark room; (b) automatic imaging. The next target is to optimally design the IP for TEM.
Acknowledgements 5. Conclusions The IP, which was developed as an image recording material for X-ray radiography, has been applied to TEM image recording. As a result, the superior properties of the IP for electrons have been ascertained. Also, its high practicability as an image recording material in TEM has been ascertained using some specimens. The IP has some advantages in its treatment and handling, in addition to its f,mdamental properties. The advantages of IP as an image recording material in TEM are listed as follows: (1) High sensitivity. (2) Wide dynamic range. (3) Variable contrast.
The authors are grateful to Dr. Y. Fujiyoshi of Protein Engineering Research Institute for his useful discussion, and to Mr. M. Hakamata of Fuji Photo Film Co., Ltd., for helping operate the reader and printer for the IP-B.
References [1] L. Reimer, Transmission Electron Microscopy (Springer, Berlin, 1984). [2] E. Zeitler, Ultramicro'vopy 10 (1982) 1. [3] Y. Fujiyoshi, T. Kobayasti, N. Uyeda, Y. Ishida and Y. Harada, Ultramicroscopy 5 (1980) 459. [4] G. Wrigley, E. Brown and P.K. Chillingworth, J. Microscopy 130 (1983) 225.
N. Mori et at / Application of "'imaging plate'" to TF,M image recording [5] M. Sonoda et ai., Radiology 148 (1983) 833, {61 S. lchihara et al., J. Electron Microsc. 33 (1984) 255. [7] N. Mori et al., in: Proc. l l t h Intern. Conf. on Electron Microscopy, Kyoto, 1986, Eds. T. lmura, S. Maruse and T. Suzuki (Japan. Soc. Electron Microscopy, Tokyo, 1986) Vol. I, p. 29. [8] T. Oikawa et al., in: Proc. l l t h Intern. Conf. on Electron Microscopy, Kyoto, 1986, Eds. T. Imura, S, Maruse and T. Suzuki (Japan. Soc. Electron Microscopy, Tokyo, 1986) Vol. 1, p. 439.
201
[9] K. Takahashi, K. Kohda, J. Miyahara, Y. Kanemitsu, K. Amitani and S. Shionoya. J. Luminescence 31 & 32 (/984) 266. [10] K. Takahashi, J. Miyahara and Y. Shibahara, J. Electrochem. Soe. 132 (1995) 1492. [111 D.J. Perry and J.G. Holt, Med. Phys. 7 (1980) 207. [12] L. Katz and A.S. Penfold, Rev. Mod. Phys. 24 (1952) 30. [131 G.W. Ludwig and J.D. Kingsley, J. Electrochem. SOc. Solid Slate Sci. 117 (1970) 348.