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High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy
ultramicroscopy
North-HollandUltramicr°sc°py 52(1993)21-29
High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy G.Y. Fan * a n d M a r k H. E l l i s m a n San Diego Microscopy and Imaging Resource, Department of Neurosciences, Uni~ersity of California at San Diego, La Jolla, CA 92093-0608, USA
Received 10 February 1993; in final form 6 July 1993
A lens-coupled slow-scan CCD camera has been built for transmission electron microscopy (TEM) applications. In this design, a leaded glass window, which is coated with a 20 p~m layer of red P20 phosphor, is mounted on the bottom of the microscope. A lens assembly and mirror prism, located outside the microscope vacuum below the leaded glass, relays the image onto a back-thinned lk× lk charge-coupled device (CCD) detector. This CCD is electronically cooled to below -30°C during operation. It is found that X-ray irradiation, generally found to be annoying in fiber-optically coupled CCD cameras, is completely eliminated by this configuration. The collection efficiency of this system, although not as high as some of the fiber-optically coupled CCD cameras, is high enough to achieve single-electron sensitivity under a high-gain mode.
I. Introduction Direct digital recording with slow-scan C C D cameras has been introduced to electron microscopy and provides a significant advantage over film such as higher sensitivity and dynamic range [1-9]. C a m e r a systems are already available in several c o n f i g u r a t i o n s (e.g., A d v a n c e d Microscopy Systems, A s t r o m e d , G a t a n and Tietz Video). T h e p e r f o r m a n c e characteristics of highquality, large-format C C D chips (1024 2 to 4096 2) challenge the d o m i n a n c e of film as the recording m e d i u m for m a n y scientific imaging requirements. These cameras are revolutionizing the way microscopists think about addressing problems of data acquisition and have b e e n used by us and by others in the analysis of specimens important to biology and materials science. These imagers, coupled with digital control of the m o d e r n electron microscope, also provide for significant ira-
* Corresponding author.
p r o v e m e n t in the control of parameters which govern the optical p e r f o r m a n c e of the instrument [10-131. Interest in the e n h a n c e m e n t of device sensitivity by fiber-optic coupling and research aimed at improving resolution and q u a n t u m yield of scintillators has oriented the research and development strategies leading to the currently available devices. To achieve the objective of producing the highest-sensitivity systems, the main development efforts and commercially available products have used fiber-optic coupling of the C C D directly to either thinned transmission Y A G or p h o s p h o r scintillator screens. T h i n n e d Y A G scintillators p r e d o m i n a t e d as the electron-to-photon conversion elements for the early devices because of their uniformity and stability. However, D a b e r k o w and coworkers [7] have d e m o n s t r a t e d that although the detection q u a n t u m efficiency ( D Q E ) of a 50 ~ m thick Y A G is at least equivalent to film at 100 kV, it drops to 0.5 at 300 kV and would be significantly worse at 400 kV. T h e use of thinner Y A G screens to improve the modulation transl%r function ( M T F ) would result in an
0304-3991/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved
22
G.Y. Fan, M.H. Eflisman / Lens-coupled slow-scan CCD camera
even poorer DQE. Realizing this impasse we have been investigating improvements in transmission phosphor screens for our implementation of a camera system for a 400 kV JEM-4000EX microscope. Similarly, several currently popular commercial designs have adopted various phosphors, multi-channel plates or scintillating fiber optics, searching for better brightness and spatial resolution [14]. X-ray irradiation of the CCD chip is a major problem associated with the application of fiberoptically coupled designs to higher-voltage TEMs. Each X-ray quantum may affect several adjoining pixels. These artifacts are frequently observed in images obtained at 400 kV with the YAG, fiberoptically coupled video-rate CCD system we currently use. Direct irradiation of the CCD by X-ray [15] and by electrons [16] can lead to degradation of the CCD, and a dose-dependent increase in the dark current has been noted as well as degradation of image fidelity. Although in some early designs [17,18], lens coupling has been used to transfer photons released in a scintillator to a sensor (e.g., silicon-intensified target), those interested in slow-scanCCD-based systems have generally dismissed the possibility of using a lens-coupled mirror system to avoid the X-ray problem because lens coupling leads to a greater loss of light than fiber-optic coupling. However, two characteristics of some of the newer CCD imagers make such lens coupling more appropriate for many applications. First, with the back-thinned (and back-side illuminated) devices, one may obtain a more than 2 x improvement in quantum efficiency. Second, a further boost of about 30% in the D Q E may be obtained by illuminating these devices with red light. To take advantage of this peak sensitivity we have characterized a red phosphor that provides good resolution and brightness in response to electrons when deposited in layers as thin as 10 p.m on leaded glass. In this paper, we describe a lens-coupled slow-scan CCD camera system for TEM applications, which avoids the problems associated with the fiber-optic coupling, and yet has a collection efficiency high enough to be single-electron sensitive.
flange
J J"-~'~'~\\""\\\\~"~'~"'"- J I Ie a d
II
~
II
shield
focusing wheel
Fig. 1. Design of a lens-coupled slow-scan CCD camera for TEM applications. The entire assembly is enclosed in a leadlined box.
2. System design The design of our camera assembly is illustrated in fig. 1. The bottom flange of the JEM4000EX microscope is replaced by a special flange which carries a large rectangular leaded glass window (130 x 100 x 20.4 mmS). This window is uniformly coated with a 20 ~ m thick layer of red P20 phosphor (YsOS:Eu, obtained from G T E / Sylannia). The phosphor is applied to the leaded glass window by a proprietary technique for maintaining a uniformly thin layer by settling, developed by Grant Scientific (Gilbert, SC, USA), after selecting grains of about 3 p.m from the bulk phosphor by a decanting method. The edges of the window are stripped off the phosphor coating to provide a suitable sealing surface for the vacuum O-ring (which seals the window to the special flange) and the entire window is sputter-coated with a thin conductive and protective layer of A u - P t . A two-lens relay is used to provide efficient coupling between the image on the phosphor scintillator and the CCD imager. An f l.0 lens (Goerz optical) with a diameter of 122 mm and an effective focal length 122 mm (front focal length = 71.6 mm) is used as the collector. For flexibility, a second lens, with a f-number of 2.0
G. Y Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
and a focal length of 100 mm, is used to form an image at the desired position. A mirror prism, of the Amici type, is used to " b e n d " the optical path by 90 ° to prevent X-ray quanta which may penetrate the leaded glass from hitting the CCD detector. Mirror prisms in general are more efficient than mirrors because of internal reflection, and the Amici type also has the property of maintaining an erect rather than " m i r r o r e d " image. With this arrangement the image may be relayed to the camera 1:1, or a demagnification factor may be selected (up to 3 : 1) by moving the lens assembly up and down, and readjusting the position of the CCD camera, mounted on a dovetail slider, to obtained a focused image. Demagnification is desired in experiments in which resolution is of concern, since the phosphor "grains" (actually it is the electron b e a m spread in phosphor which limits the resolution) can be demagnifled to reduce the influence of the scintillator resolution on the overall system resolution. However, demagnifying the phosphor image means moving the lens assembly away from the scintillator thereby reducing the collection efficiency. Thus in experiments which employ minimum-dose image acquisition methods, a 1:1 image relay factor should be used. Focusing adjustments to set up the camera system may be made manually while operating the microscope up to 200 kV as the leaded glass window provides sufficient X-ray attenuation until excitation exceeds this level. As an additional convenience, the CCD camera head (Photometrics, CH250) can be rotated relative to the rest of the assembly so that a particular axis on the image can be aligned with respect to the computer screen. This is important, for example, in stereo-pair and tomographic series acquisition where the tilting axis of the specimen holder should be parallel to a row or column of the CCD, and to facilitate the collection of large-format images by montaging [19]. Such montaging is being accomplished in this laboratory by systematically shifting the image relative to CCD by changing the x and y projector deflector excitation. The camera rotation is driven by a servo motor coupled with encoder and can be digitally
23
controlled so that the CCD orientation can be adjusted easily and precisely. As noted above, the leaded glass can effectively shield X-ray up to 200 kV. The CCD detector itself is free from X-ray at much higher voltages because of the 90 ° optical path change as well as a lead "baffle" plate just above the camera head. However, above 200 kV additional shielding is needed to provide a safe environment for the operator. This additional shielding is provided by a lead-lined box which provides for complete enclosure of the camera system. With this configuration, no evidence of X-ray excitation of the CCD imager has been found in the images acquired at 400 kV. The Tektronix CCD detector used has 1024 × 1024 pixels, and is back-thinned, enhanced and A R coated, with a pixel size of 24 × 2 4 / z m 2. The CCD is operated in the backside-illumination mode, with a maximum quantum efficiency of ~ 80% in the 600-650 nm wavelength range. This sensitivity is more than double the maximum efficiency of the standard front-side-illuminated CCD used i n fiber-optically coupled cameras ( < 40%, Tektronix CCD data sheet). The particular grade 1 chip used in this camera was handselected by Tektronix, and has no defects. The CCD chip was packaged by Photometrics in a vacuum-sealed CH250 camera head with a 14-bit A / D converter running at 200 kHz (CE200A). The full well capacity of this chip was measured by both Tektronix and Photometrics to be 576 000 CCD electrons (electrons accumulated in the CCD well, not to be confused with the high-energy electrons in the microscope). The base gain of the A / D converter is set such that a number of 35.2 CCD electrons is converted to one A / D unit ( A D U ) so that the 14-bit A / D limit corresponds to the full well capacity, with a readout noise of 22 CCD e , which is less than one ADU. A high gain mode is also supported which is 4 x the base gain, with a conversion of 8.8 CCD e / A D U and a readout noise of 15 CCD e - . 3. Scintillator selection
There are mainly two reasons for employing phosphor ~nstead of Y A G as the scintillator for
24
G. E Fan, M.H. Ellisman / Lens-coupled sh)w-scan CCD camera
this camera system: resolution and light yield. Although the single-crystal Y A G scintillators (see, e.g., ref. [20] for a review) generally have a more uniform light emission than polycrystalline phosphor [7], they are difficult to thin to a uniform thickness to achieve a resolution of ~ 30 /zm. It is noteworthy that the resolution is determined, largely, by the electron spread in the scintillator which has the same order of magnitude as the
thickness (within penetration range). Daberkow and coworkers [7] have reported a resolution of 62 and 84 p.m with 100 and 300 keV electrons, respectively, from a YAG screen of 50 /~m in thickness. Clearly, unless there is a substantial demagnification of the scintillating screen, the point-spread function will cover several CCD pixels thereby effectively reducing the number of pixe[s in an image. This is in contrast to transmis-
Fig. 2. Images of a 0.25 ~ m section of myelin from the mouse peripheral nervous system. Exposures are 0.1, 1, 10 and 100 s for (a), (b), (c) and (d), corresponding to an incident dose of 0.45, 4.5, 45 and 450 e / p i x e l on CCD, respectively, with the same illumination conditions.
G.Y. Fan, M,H. Ellisman /Lens-coupled slow-scan CCD camera
sion phosphor screens which can be fabricated with a uniform phosphor layer as thin as 5 /~m deposited on the leaded glass or other flat substrates. Such scintillators can be expected to provide considerably better resolution. A M T F value as high as 0.28 at 50 line p a i r s / m m (or 20 /xm spacing) has been reported from a 5 p+m thick layer of phosphor deposited on glass (measured in a SEM, voltage unspecified) [21]. In addition to the potential for improved resolution, the light yield of phosphor is generally a few times greater than that from Y A G of the same thickness [22]. The P20 phosphor (Y3OS:Eu) we chose for this design generates light with wave length sharply peaked at 625 nm. This was chosen to match the sensitivity characteristics of the backthinned Tektronix CCD imager, as the quantumefficiency peaks between 600-650 nm. This provides this system with a boost in quantum efficiency of about 30% when compared to that which would be obtained with the green light (560 nm) generated by a Y A G scintillator.
4. Experimental images The flexibility offered by the lens coupling allows the system to be configured in different ways to meet specific experimental needs. The experimental images (fig. 2) included here were collected with the low dose 1 : 1 lens relay configuration. The theoretical collection efficiency of this arrangement can be as high as 6.2%, but reflection losses and f - n u m b e r mismatch of the second lens ( f 2 , focal length = 100 mm) reduce collection efficiency to an estimated value of 3%. Fig. 2 shows the images of a 0.25 /xm section of mouse peripheral nerve myelinated axons, taken with the above-described lens configuration at 200 kV and a microscope magnification of 40 000 × . These images were acquired under identical conditions except that exposure duration was varied over a range of three orders of magnitude from 0.1, 1, 10 to 100 seconds. The mean intensity versus exposure time of these images is plotted in fig. 3, from which one can see that the linearity of the system is excellent in the exposure range covering three orders of magnitude. The
25
10000~
I 000 +-,
g
o (J v >~ 100" c: c
10
1
. . . . . . . . .1
, 1
. . . . . . . .
, 10
. . . . . . . 100
Exposure Time (s) Fig. 3. Mean intensity of the images in fig. 2, showing the linearity of the C C D camera with exposure covering three orders of magnitude.
electron conversion in this configuration is approximately one primary electron to 12 A D U and is calibrated as follows. The magnification of the microscope is first set to 4000 x , and the illumination is adjusted so that a current reading 18 p A / c m ~ is obtained with the microscope focus screen. This illumination level translates to 7.8 × 10 7 e s-1 cm 2 on the phosphor scintillator also taking into consideration the fact that the scintillator is about 20% lower than the focus screen from the last beam crossover. The microscope magnification is then increased 10-fold to 40000 x , reducing the illumination on the scintillator by a factor of100, so that 7.8 ×105 e - s l c m 2 is achieved, or equivalently, 4.5 e - s 1 pixel t on the CCD detector (pixel size = 2 4 / z m × 24/xm). At this level of illumination the current on the focus screen is below the detection limit, and the image is not visible by the unaided eye. With an exposure time of one second, the image in fig. 2b has a mean of 53.04, which gives the overall conversion of one primary electron to 12 A D U in the image. Fig. 2a is acquired with an exposure time of 0.1 s, equivalent to a dose of 0.45 e - / p i x e l on the CCD detector and 0.018 e - / ~ 2 on the specimen (neglecting electrons stopped by the objective
26
G, Y. Fan, M.H. Ellisman / Lens-coupled slow-scan CCD camera
a p e r t u r e ) . T h e fact that the myelin p a t t e r n is d i s c e r n i b l e in this i m a g e with an a v e r a g e of less t h a n o n e p r i m a r y e l e c t r o n p e r pixel indicates t h a t the c a m e r a in this lens c o n f i g u r a t i o n is singlee l e c t r o n sensitive, a n d is suitable for g e n e r a l low-dose work. F u r t h e r i n c r e a s e in sensitivity will unjustifiably r e d u c e d the d y n a m i c r a n g e of the system. In fact, one w o u l d n o r m a l l y w a n t to reduce the overall gain of the system to 1 or m o r e p r i m a r y e l e c t r o n s p e r A D U to maximize the dyn a m i c range of the system.
5. O t h e r c h a r a c t e r i s t i c s
P20 red pho,sphor screen
Efficiency Energy transferred Refraction index Photon wavelength Photon energy
E
0.2
AE n0 A LTph
1.2 625 nm 1.98 eV
Lens system
Lens transfer efficiency Lens magnification Focal length F-number Geometric distortion
M f l , f2 I:1, F2
0.03 < 1, variable 122 mm, 100 mm 1,2 < 0.2%
Back-thinned TK1024 CCD (at - 35 °) ~7('¢'D 0.8 de(. D 24/~m
5.1, D O E
T h e D Q E of the system can be c a l c u l a t e d b a s e d on t h e f o r m u l a d e r i v e d by H e r r m a n n a n d Liu [23] for a l e n s - c o u p l e d system. T h e f o r m u l a is d u p l i c a t e d h e r e for c o n v e n i e n c e , with exactly the same notation: D Q E = [1 + v a r A E / ( A E )
Table 1 System parameters
2
Quantum efficiency Pixel size Pixel number Full-well capacity Dark current Readout-noise Conversion factor Readout speed A / D precision Storage time Primary e per pixel
D ?lr
t N
1024 X 1024 5.76 × 105 e 16.4 e s i pixel i 22 and 15 e /pixel 35.2 and 8.8 e /ADU 20(1 kHz 14 bit variable variable
+ (1 - "qLr/CCD)/(ne) +N-l(n~+Dr)/(n~)
2]
',
(i)
we o b t a i n D Q E = 0.71 for N = 1 and D Q E = 0.72 for N > 10.
where ( n ~ ) = e( A E / E p h ) 7 1 L ~ C C D
(2)
is the m e a n n u m b e r of e l e c t r o n - h o l e p a i r s g e n e r a t e d in the C C D by one p r i m a r y electron. T h e d e f i n i t i o n s and values of t h e p a r a m e t e r s are p r e s e n t e d in table 1. Since o n e p r i m a r y e is c o n v e r t e d to 12 A D U (section 4) a n d o n e A D U c o r r e s p o n d s to 8.8 e l e c t r o n - h o l e pairs in the C C D (section 2), we have ( n ~ ) = 105.6 (at 200 kV), thus the last two t e r m s in eq. (1) a r e small even at low dose, e.g., for N = 1. H e n c e t h e D Q E of this system is d o m i n a t e d by the statistics of the e l e c t r o n - p h o t o n conversion process. If we neglect the last two t e r m s for now, t h e n a r e c e n t calculation by Kujawa a n d Krahl [8] for P20 phosp h o r at 100 kV l e a d s to a D Q E of 0.72. T a k i n g into account the last two t e r m s with the values listed in table 1, a n d the fact that this p h o s p h o r is 70% m o r e efficient at 100 kV, so ( n ~ ) = 179.5,
5.2. G e o m e t r i c distortion
T h e two c o m p o u n d lenses u s e d in the relay a r e of e x c e p t i o n a l l y high quality. T h e g e o m e t r i c dist o r t i o n of the system was m e a s u r e d on an optical b e n c h using a sheet of g r a p h p a p e r consisting of 1 m m s q u a r e grids as the object. T h e d i s t o r t i o n is less t h a n 0.2% over the w h o l e field of view ( ~ 24 × 24 mm2). N o b e n d i n g of the lines can be det e c t e d even n e a r the e d g e [24]. 5.3. S h a d i n g distortion
T h e d a r k c u r r e n t of t h e C C D is fairly flat, with the e x c e p t i o n of o n e c o l u m n which has a h i g h e r value of 8 A D U t h a n the rest C C D pixels b u t shows no a b n o r m a l gain v a r i a t i o n w h e n t h e r e is a d e q u a t e illumination, e.g., N > 10, a n d t h e r e fore, is not c o n s i d e r e d a d e f e c t by the m a n u f a c -
G.Y. Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
turer. For low-dose work, it is necessary to correct this by subtracting the bias pattern from the image. The vignetting of the lens system introduces some shading distortion (multiplicative), causing the periphery of an image to be darker than the center. There is about 17% variation from corner to center with uniform illumination. Also, the phosphor grains are visible with uniform illumination. These shading distortions do not significantly distract on-line observation, but the final images should be corrected by a simple gain-normalization process, described by many authors, e.g. refs. [7,8]. With a pixel depth of 14 bits, at least 12 bits can be retained after the gain-normalization process. The images in fig. 2 have been corrected for both additive and multi° plicative shading distortions.
5.4. Resolution The resolution of the phosphor layer was measured using the edge test method. To avoid scratching the phosphor coating of the working screen, the m e a s u r e m e n t was performed on a similarly coated test screen (3 m m thick glass disk 34 m m in diameter). In order to obtain a good accuracy in the measurement, a different lens combination was used providing a magnification of 3.4 x with a CCD camera head (Photometrics CH250) of smaller CCD pixel size (6 x 6 ~ m 2, Kodak KAF1400). The full-width-half-maximum ( F W H M ) of the differential of the knife edge, averaged over 100 lines, was found to be 33 p~m _+ 10% at 200 kV. The M T F of the system was estimated from the power spectrum of images of uniform illumination [25], and the result is in agreement with the phosphor resolution measurement. So the total number of effective pixels is less than 1024 x 1024 with the 1:1 imaging configuration. A demagnification factor of 2 is preferable in order to fully utilize the CCD pixels, at the cost of reduced sensitivity.
5.5. Glare in the lens assembly The interior of the cylinder housing of the lens assembly has been coated with non-reflecting black paint to minimize stray light. However,
27
internal reflections and diffuse scattering cannot be completely eliminated in a lens system. Optical bench tests of this system with a 10 p~m pin-hole as the object and a diffuse light source revealed a background below 0.06% and a halo around the pin-hole about 0.4% of the maximum intensity measured within the pin-hole image. Analysis of T E M diffraction patterns (with a 4 /zm objective aperture) acquired using this system indicated a lower value for the background of 0.03% and a halo of 0.2%. Presumably the lower values obtained in the T E M test are due to the fact that the phosphor screen is a self-illuminating object. Therefore, the final image does not suffer from interference of the blurred image of the light source which would otherwise be formed by the pin-hole. These values indicate that reflections and diffuse scattering in the lens system is not a serious problem for most T E M imaging and diffraction applications.
6. Discussion
Lens coupling has several advantages over the fiber-optic coupling based on authors' experience with both types of coupling schemes. No X-ray irradiation: In a fiber-optically coupled system, X-ray quanta generated in the microscope as well as those generated by electrons striking the scintillator go through the fiber-optic plate and strike the CCD chip, creating bright spots on the image. A group of typically 3 x 3 pixels or less can be affected by one X-ray quantum. The intensity of these spots varies greatly, ranging from just above the dark noise level to saturation of the CCD pixels. The number of these bright spots in an image depends on the high voltage used and integration time. These artifacts can be fixed to some degree by post image-processing, e.g., by replacing the group of pixels with the mean of its surrounding pixels. Another solution would be to record the same image 3 times and take the median of each corresponding pixel to form a new image. Obviously this is not dose efficient and requires that specimen drift is small. The use of longer and leaded fiber-optic rods should lessen the problem, but at
28
G.Y. Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
the cost of reduced light transmittance. In contrast, this problem is eliminated in our camera system by "bending" the optical path 90 ° and placing a lead-baffle plate above the camera head, No interference with the microscope t,acuum system: Since the microscope vacuum is sealed by a leaded glass window in our camera and the rest of the system is outside of the microscope, the camera operation as well as reconfiguration do not interfere the microscope vacuum. This is an improvement over most fiber-optically coupled systems where the entire CCD-fiber-optic coupling unit is cooled and is inside the microscope vacuum. The unit acts as a cold trap in the portion of the column maintained with the poorest vacuum and thus requires periodic cleaning. In addition, the units must be protected from condensation and ice formation which would result from venting of the camera chamber area of the microscope. No honey-comb patterns: The honey-comb pattern in a f!ber-optically coupled system, although correctable by the gain-normalization process, interferes with on-line observation where computational speed may not permit on-line gain normalization. The lens-coupled system is free from this type of pattern. Easy to modify or expand." The modular design and flexibility of the lens coupling allows the system to be reconfigured easily. Custom modification or expansion of a fiber-optically coupled system is not so convenient because of its rigid coupling to the fragile and expensive CCD chip. While lens coupling provides the above-mentioned advantages, it suffers two drawbacks when compared with the fiber-optic coupling. The system requires more space and is lower in overall collection efficiency. Further improvement of the collection efficiency by using larger lenses will result in a slightly larger apparatus. The bulkier size does reduce the leg room in the knee space of the microscope but this does not appear to inconvenience microscope operators. 7. Conclusions We have demonstrated that, by choosing proper lens combinations and matching phosphor
scintillator material characteristics to the spectral sensitivity of the CCD imagery it is possible to achieve single-electron detection with a lens-coupled slow-scan CCD camera while still retaining a reasonably large dynamic range. Although the collection efficiency of a few percent of the lens system does not quite match that of a typical fiber-optically coupled CCD system (10%-20%), this does not represent a fundamental limitation. The flexibility and other advantages gained with the lens coupling far outweigh this shortcoming and afford the slow-scan CCD camera a better coupling for T E M applications.
Acknowledgments We thank referee K.-H. H e r r m a n n and Editor P. Kruit for their critical review and comments. We are grateful to Stephen Young and Ronald Milligan for helpful discussions. Assistance from Tektronix, Photometrics and Grant Scientific is gratefully acknowledged. This project was supported by NSF grant DCB 8811713, N I H grants HL27470, NS14718, NS26739, RR04050 and a grant from the H u m a n Frontiers of Science to M.H.E.
References [1] M.E. Mochel and J.M. Mochel, in: Proc. 44th Annual EMSA Meeting, 1986, p. 616. [2] G.Y. Fan and J.H. Butler, in: Computer Simulation of Electron Microscope Diffraction and Images, Eds. W. Krakow and M. O'Keefe (The Minerals, Metals and Materials Society, Warrendale, PA, 1989) p. 195. [3] R.S. Aikens, D.A. Agard and J.W. Sedat, Meth. Cell Biol. 29 (1989) 291. [4] J.C.H. Spence and J.M. Zuo, Rev. Sci. Instr. 59(9)(1988) 2102. [5] P.E. Mooney, G.Y. Fan, C.E. Meyer, K.V. Troung, D.B. Bui and O.L. Krivanek, in: Proc. 12th Int. Congr. for Electron Microscopy, 1990, p. 164. [6] J.F. Mancuso, W.B. Maxwell, R.E. Camp and M.H. Ellisman, in: Proc. 50th Annual EMSA Meeting, Vol. 2, 1992, p. 946. [7] 1. Daberkow, K.-H. Herrmann, L. Liu and W.D. Rau, Ultramicroscopy 38 (1991) 215. [8] S. Kujawa and D. Krahl, Ultramicroscopy 46 (1992) 395. [9] O.L. Krivanek and P.E. Mooney, UItramicroscopy 49 (1992) 95.
G. ]I.. Fan, M.H. Ellisman / L e n s - c o u p l e d slow-scan CCD camera
[10] G.Y. Fan and O.L. Krivanek, in: Proc. 12th Int. Congr. for Electron Microscopy, 1990, p. 532. [11] K. Dierksen, D. Typke, R. Hegerl, A.J. Koster and W. Baumeister, Ultramicroscopy 40 (1992) 71. [12] D.A. Agard, A.J. Koster, M.B. Braunfeld and J.W. Sedat, in: Proc. 50th Annual EMSA Meeting, 1992, p. 1044. [13] G.Y. Fan, P.E. Mooney, W.J. DeRuijter and O.L. Krivanek, Proc. Jpn. Electron Microsc. Soc. (1992) 128. [14] O.L. Krivanek, P.E. Mooney and G.Y. Fan, Inst. Phys. Conf. Set. 119 (1991) 523. [15] R.H. Dyck, LORAL/Fairchild Imaging Sensors Catalog (1992). [16] P.T.E. Roberts, J.N. Chapman and A.M. MacLeod, UItramicroscopy 8 (1982) 385. [17] R. Valle, B. Genty, A. Marraud and B. Bardo, in: Proc. 6th Eur. Conf. on Electron Microscopy, 1976, p. 336.
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[18] H. Shuman, C.F. Chang and A.P. Somlyo, Ultramicroscopy 19 (1986) 121. [19] G.Y. Fan, A.J. Gubbens, O.L. Krivanek, M.L. Leber and P.E. Mooney, in: Proc. 49th Annual EMSA Meeting, 1991, p. 524. [20] J.N. Chapman, A.J. Craven and C.P. Scott, Ultramicroscopy 28 (1989) 108. I21] A.J. Jenkins, Assessment of Imaging Systems: Visible and Infrared 274 (1981) 154. [22] K. Yoshida, A. Takaoka, K. Ura, T. Katsuta and 1. Matsui, Ultramicroscopy 39 (1991) 45. [23] K.-H. Herrmann and L. Liu, Optik 92(2) (1992) 48. [24] G.Y. Fan and M.H. Ellisman, in: Proc. 51st Annual MSA Meeting, 1993, p. 642. [25] W.J. de Ruijter and J.K. Weiss, Rev. Sci. Instr. 63 (1992) 4314.
North-HollandUltramicr°sc°py 52(1993)21-29
High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy G.Y. Fan * a n d M a r k H. E l l i s m a n San Diego Microscopy and Imaging Resource, Department of Neurosciences, Uni~ersity of California at San Diego, La Jolla, CA 92093-0608, USA
Received 10 February 1993; in final form 6 July 1993
A lens-coupled slow-scan CCD camera has been built for transmission electron microscopy (TEM) applications. In this design, a leaded glass window, which is coated with a 20 p~m layer of red P20 phosphor, is mounted on the bottom of the microscope. A lens assembly and mirror prism, located outside the microscope vacuum below the leaded glass, relays the image onto a back-thinned lk× lk charge-coupled device (CCD) detector. This CCD is electronically cooled to below -30°C during operation. It is found that X-ray irradiation, generally found to be annoying in fiber-optically coupled CCD cameras, is completely eliminated by this configuration. The collection efficiency of this system, although not as high as some of the fiber-optically coupled CCD cameras, is high enough to achieve single-electron sensitivity under a high-gain mode.
I. Introduction Direct digital recording with slow-scan C C D cameras has been introduced to electron microscopy and provides a significant advantage over film such as higher sensitivity and dynamic range [1-9]. C a m e r a systems are already available in several c o n f i g u r a t i o n s (e.g., A d v a n c e d Microscopy Systems, A s t r o m e d , G a t a n and Tietz Video). T h e p e r f o r m a n c e characteristics of highquality, large-format C C D chips (1024 2 to 4096 2) challenge the d o m i n a n c e of film as the recording m e d i u m for m a n y scientific imaging requirements. These cameras are revolutionizing the way microscopists think about addressing problems of data acquisition and have b e e n used by us and by others in the analysis of specimens important to biology and materials science. These imagers, coupled with digital control of the m o d e r n electron microscope, also provide for significant ira-
* Corresponding author.
p r o v e m e n t in the control of parameters which govern the optical p e r f o r m a n c e of the instrument [10-131. Interest in the e n h a n c e m e n t of device sensitivity by fiber-optic coupling and research aimed at improving resolution and q u a n t u m yield of scintillators has oriented the research and development strategies leading to the currently available devices. To achieve the objective of producing the highest-sensitivity systems, the main development efforts and commercially available products have used fiber-optic coupling of the C C D directly to either thinned transmission Y A G or p h o s p h o r scintillator screens. T h i n n e d Y A G scintillators p r e d o m i n a t e d as the electron-to-photon conversion elements for the early devices because of their uniformity and stability. However, D a b e r k o w and coworkers [7] have d e m o n s t r a t e d that although the detection q u a n t u m efficiency ( D Q E ) of a 50 ~ m thick Y A G is at least equivalent to film at 100 kV, it drops to 0.5 at 300 kV and would be significantly worse at 400 kV. T h e use of thinner Y A G screens to improve the modulation transl%r function ( M T F ) would result in an
0304-3991/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved
22
G.Y. Fan, M.H. Eflisman / Lens-coupled slow-scan CCD camera
even poorer DQE. Realizing this impasse we have been investigating improvements in transmission phosphor screens for our implementation of a camera system for a 400 kV JEM-4000EX microscope. Similarly, several currently popular commercial designs have adopted various phosphors, multi-channel plates or scintillating fiber optics, searching for better brightness and spatial resolution [14]. X-ray irradiation of the CCD chip is a major problem associated with the application of fiberoptically coupled designs to higher-voltage TEMs. Each X-ray quantum may affect several adjoining pixels. These artifacts are frequently observed in images obtained at 400 kV with the YAG, fiberoptically coupled video-rate CCD system we currently use. Direct irradiation of the CCD by X-ray [15] and by electrons [16] can lead to degradation of the CCD, and a dose-dependent increase in the dark current has been noted as well as degradation of image fidelity. Although in some early designs [17,18], lens coupling has been used to transfer photons released in a scintillator to a sensor (e.g., silicon-intensified target), those interested in slow-scanCCD-based systems have generally dismissed the possibility of using a lens-coupled mirror system to avoid the X-ray problem because lens coupling leads to a greater loss of light than fiber-optic coupling. However, two characteristics of some of the newer CCD imagers make such lens coupling more appropriate for many applications. First, with the back-thinned (and back-side illuminated) devices, one may obtain a more than 2 x improvement in quantum efficiency. Second, a further boost of about 30% in the D Q E may be obtained by illuminating these devices with red light. To take advantage of this peak sensitivity we have characterized a red phosphor that provides good resolution and brightness in response to electrons when deposited in layers as thin as 10 p.m on leaded glass. In this paper, we describe a lens-coupled slow-scan CCD camera system for TEM applications, which avoids the problems associated with the fiber-optic coupling, and yet has a collection efficiency high enough to be single-electron sensitive.
flange
J J"-~'~'~\\""\\\\~"~'~"'"- J I Ie a d
II
~
II
shield
focusing wheel
Fig. 1. Design of a lens-coupled slow-scan CCD camera for TEM applications. The entire assembly is enclosed in a leadlined box.
2. System design The design of our camera assembly is illustrated in fig. 1. The bottom flange of the JEM4000EX microscope is replaced by a special flange which carries a large rectangular leaded glass window (130 x 100 x 20.4 mmS). This window is uniformly coated with a 20 ~ m thick layer of red P20 phosphor (YsOS:Eu, obtained from G T E / Sylannia). The phosphor is applied to the leaded glass window by a proprietary technique for maintaining a uniformly thin layer by settling, developed by Grant Scientific (Gilbert, SC, USA), after selecting grains of about 3 p.m from the bulk phosphor by a decanting method. The edges of the window are stripped off the phosphor coating to provide a suitable sealing surface for the vacuum O-ring (which seals the window to the special flange) and the entire window is sputter-coated with a thin conductive and protective layer of A u - P t . A two-lens relay is used to provide efficient coupling between the image on the phosphor scintillator and the CCD imager. An f l.0 lens (Goerz optical) with a diameter of 122 mm and an effective focal length 122 mm (front focal length = 71.6 mm) is used as the collector. For flexibility, a second lens, with a f-number of 2.0
G. Y Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
and a focal length of 100 mm, is used to form an image at the desired position. A mirror prism, of the Amici type, is used to " b e n d " the optical path by 90 ° to prevent X-ray quanta which may penetrate the leaded glass from hitting the CCD detector. Mirror prisms in general are more efficient than mirrors because of internal reflection, and the Amici type also has the property of maintaining an erect rather than " m i r r o r e d " image. With this arrangement the image may be relayed to the camera 1:1, or a demagnification factor may be selected (up to 3 : 1) by moving the lens assembly up and down, and readjusting the position of the CCD camera, mounted on a dovetail slider, to obtained a focused image. Demagnification is desired in experiments in which resolution is of concern, since the phosphor "grains" (actually it is the electron b e a m spread in phosphor which limits the resolution) can be demagnifled to reduce the influence of the scintillator resolution on the overall system resolution. However, demagnifying the phosphor image means moving the lens assembly away from the scintillator thereby reducing the collection efficiency. Thus in experiments which employ minimum-dose image acquisition methods, a 1:1 image relay factor should be used. Focusing adjustments to set up the camera system may be made manually while operating the microscope up to 200 kV as the leaded glass window provides sufficient X-ray attenuation until excitation exceeds this level. As an additional convenience, the CCD camera head (Photometrics, CH250) can be rotated relative to the rest of the assembly so that a particular axis on the image can be aligned with respect to the computer screen. This is important, for example, in stereo-pair and tomographic series acquisition where the tilting axis of the specimen holder should be parallel to a row or column of the CCD, and to facilitate the collection of large-format images by montaging [19]. Such montaging is being accomplished in this laboratory by systematically shifting the image relative to CCD by changing the x and y projector deflector excitation. The camera rotation is driven by a servo motor coupled with encoder and can be digitally
23
controlled so that the CCD orientation can be adjusted easily and precisely. As noted above, the leaded glass can effectively shield X-ray up to 200 kV. The CCD detector itself is free from X-ray at much higher voltages because of the 90 ° optical path change as well as a lead "baffle" plate just above the camera head. However, above 200 kV additional shielding is needed to provide a safe environment for the operator. This additional shielding is provided by a lead-lined box which provides for complete enclosure of the camera system. With this configuration, no evidence of X-ray excitation of the CCD imager has been found in the images acquired at 400 kV. The Tektronix CCD detector used has 1024 × 1024 pixels, and is back-thinned, enhanced and A R coated, with a pixel size of 24 × 2 4 / z m 2. The CCD is operated in the backside-illumination mode, with a maximum quantum efficiency of ~ 80% in the 600-650 nm wavelength range. This sensitivity is more than double the maximum efficiency of the standard front-side-illuminated CCD used i n fiber-optically coupled cameras ( < 40%, Tektronix CCD data sheet). The particular grade 1 chip used in this camera was handselected by Tektronix, and has no defects. The CCD chip was packaged by Photometrics in a vacuum-sealed CH250 camera head with a 14-bit A / D converter running at 200 kHz (CE200A). The full well capacity of this chip was measured by both Tektronix and Photometrics to be 576 000 CCD electrons (electrons accumulated in the CCD well, not to be confused with the high-energy electrons in the microscope). The base gain of the A / D converter is set such that a number of 35.2 CCD electrons is converted to one A / D unit ( A D U ) so that the 14-bit A / D limit corresponds to the full well capacity, with a readout noise of 22 CCD e , which is less than one ADU. A high gain mode is also supported which is 4 x the base gain, with a conversion of 8.8 CCD e / A D U and a readout noise of 15 CCD e - . 3. Scintillator selection
There are mainly two reasons for employing phosphor ~nstead of Y A G as the scintillator for
24
G. E Fan, M.H. Ellisman / Lens-coupled sh)w-scan CCD camera
this camera system: resolution and light yield. Although the single-crystal Y A G scintillators (see, e.g., ref. [20] for a review) generally have a more uniform light emission than polycrystalline phosphor [7], they are difficult to thin to a uniform thickness to achieve a resolution of ~ 30 /zm. It is noteworthy that the resolution is determined, largely, by the electron spread in the scintillator which has the same order of magnitude as the
thickness (within penetration range). Daberkow and coworkers [7] have reported a resolution of 62 and 84 p.m with 100 and 300 keV electrons, respectively, from a YAG screen of 50 /~m in thickness. Clearly, unless there is a substantial demagnification of the scintillating screen, the point-spread function will cover several CCD pixels thereby effectively reducing the number of pixe[s in an image. This is in contrast to transmis-
Fig. 2. Images of a 0.25 ~ m section of myelin from the mouse peripheral nervous system. Exposures are 0.1, 1, 10 and 100 s for (a), (b), (c) and (d), corresponding to an incident dose of 0.45, 4.5, 45 and 450 e / p i x e l on CCD, respectively, with the same illumination conditions.
G.Y. Fan, M,H. Ellisman /Lens-coupled slow-scan CCD camera
sion phosphor screens which can be fabricated with a uniform phosphor layer as thin as 5 /~m deposited on the leaded glass or other flat substrates. Such scintillators can be expected to provide considerably better resolution. A M T F value as high as 0.28 at 50 line p a i r s / m m (or 20 /xm spacing) has been reported from a 5 p+m thick layer of phosphor deposited on glass (measured in a SEM, voltage unspecified) [21]. In addition to the potential for improved resolution, the light yield of phosphor is generally a few times greater than that from Y A G of the same thickness [22]. The P20 phosphor (Y3OS:Eu) we chose for this design generates light with wave length sharply peaked at 625 nm. This was chosen to match the sensitivity characteristics of the backthinned Tektronix CCD imager, as the quantumefficiency peaks between 600-650 nm. This provides this system with a boost in quantum efficiency of about 30% when compared to that which would be obtained with the green light (560 nm) generated by a Y A G scintillator.
4. Experimental images The flexibility offered by the lens coupling allows the system to be configured in different ways to meet specific experimental needs. The experimental images (fig. 2) included here were collected with the low dose 1 : 1 lens relay configuration. The theoretical collection efficiency of this arrangement can be as high as 6.2%, but reflection losses and f - n u m b e r mismatch of the second lens ( f 2 , focal length = 100 mm) reduce collection efficiency to an estimated value of 3%. Fig. 2 shows the images of a 0.25 /xm section of mouse peripheral nerve myelinated axons, taken with the above-described lens configuration at 200 kV and a microscope magnification of 40 000 × . These images were acquired under identical conditions except that exposure duration was varied over a range of three orders of magnitude from 0.1, 1, 10 to 100 seconds. The mean intensity versus exposure time of these images is plotted in fig. 3, from which one can see that the linearity of the system is excellent in the exposure range covering three orders of magnitude. The
25
10000~
I 000 +-,
g
o (J v >~ 100" c: c
10
1
. . . . . . . . .1
, 1
. . . . . . . .
, 10
. . . . . . . 100
Exposure Time (s) Fig. 3. Mean intensity of the images in fig. 2, showing the linearity of the C C D camera with exposure covering three orders of magnitude.
electron conversion in this configuration is approximately one primary electron to 12 A D U and is calibrated as follows. The magnification of the microscope is first set to 4000 x , and the illumination is adjusted so that a current reading 18 p A / c m ~ is obtained with the microscope focus screen. This illumination level translates to 7.8 × 10 7 e s-1 cm 2 on the phosphor scintillator also taking into consideration the fact that the scintillator is about 20% lower than the focus screen from the last beam crossover. The microscope magnification is then increased 10-fold to 40000 x , reducing the illumination on the scintillator by a factor of100, so that 7.8 ×105 e - s l c m 2 is achieved, or equivalently, 4.5 e - s 1 pixel t on the CCD detector (pixel size = 2 4 / z m × 24/xm). At this level of illumination the current on the focus screen is below the detection limit, and the image is not visible by the unaided eye. With an exposure time of one second, the image in fig. 2b has a mean of 53.04, which gives the overall conversion of one primary electron to 12 A D U in the image. Fig. 2a is acquired with an exposure time of 0.1 s, equivalent to a dose of 0.45 e - / p i x e l on the CCD detector and 0.018 e - / ~ 2 on the specimen (neglecting electrons stopped by the objective
26
G, Y. Fan, M.H. Ellisman / Lens-coupled slow-scan CCD camera
a p e r t u r e ) . T h e fact that the myelin p a t t e r n is d i s c e r n i b l e in this i m a g e with an a v e r a g e of less t h a n o n e p r i m a r y e l e c t r o n p e r pixel indicates t h a t the c a m e r a in this lens c o n f i g u r a t i o n is singlee l e c t r o n sensitive, a n d is suitable for g e n e r a l low-dose work. F u r t h e r i n c r e a s e in sensitivity will unjustifiably r e d u c e d the d y n a m i c r a n g e of the system. In fact, one w o u l d n o r m a l l y w a n t to reduce the overall gain of the system to 1 or m o r e p r i m a r y e l e c t r o n s p e r A D U to maximize the dyn a m i c range of the system.
5. O t h e r c h a r a c t e r i s t i c s
P20 red pho,sphor screen
Efficiency Energy transferred Refraction index Photon wavelength Photon energy
E
0.2
AE n0 A LTph
1.2 625 nm 1.98 eV
Lens system
Lens transfer efficiency Lens magnification Focal length F-number Geometric distortion
M f l , f2 I:1, F2
0.03 < 1, variable 122 mm, 100 mm 1,2 < 0.2%
Back-thinned TK1024 CCD (at - 35 °) ~7('¢'D 0.8 de(. D 24/~m
5.1, D O E
T h e D Q E of the system can be c a l c u l a t e d b a s e d on t h e f o r m u l a d e r i v e d by H e r r m a n n a n d Liu [23] for a l e n s - c o u p l e d system. T h e f o r m u l a is d u p l i c a t e d h e r e for c o n v e n i e n c e , with exactly the same notation: D Q E = [1 + v a r A E / ( A E )
Table 1 System parameters
2
Quantum efficiency Pixel size Pixel number Full-well capacity Dark current Readout-noise Conversion factor Readout speed A / D precision Storage time Primary e per pixel
D ?lr
t N
1024 X 1024 5.76 × 105 e 16.4 e s i pixel i 22 and 15 e /pixel 35.2 and 8.8 e /ADU 20(1 kHz 14 bit variable variable
+ (1 - "qLr/CCD)/(ne) +N-l(n~+Dr)/(n~)
2]
',
(i)
we o b t a i n D Q E = 0.71 for N = 1 and D Q E = 0.72 for N > 10.
where ( n ~ ) = e( A E / E p h ) 7 1 L ~ C C D
(2)
is the m e a n n u m b e r of e l e c t r o n - h o l e p a i r s g e n e r a t e d in the C C D by one p r i m a r y electron. T h e d e f i n i t i o n s and values of t h e p a r a m e t e r s are p r e s e n t e d in table 1. Since o n e p r i m a r y e is c o n v e r t e d to 12 A D U (section 4) a n d o n e A D U c o r r e s p o n d s to 8.8 e l e c t r o n - h o l e pairs in the C C D (section 2), we have ( n ~ ) = 105.6 (at 200 kV), thus the last two t e r m s in eq. (1) a r e small even at low dose, e.g., for N = 1. H e n c e t h e D Q E of this system is d o m i n a t e d by the statistics of the e l e c t r o n - p h o t o n conversion process. If we neglect the last two t e r m s for now, t h e n a r e c e n t calculation by Kujawa a n d Krahl [8] for P20 phosp h o r at 100 kV l e a d s to a D Q E of 0.72. T a k i n g into account the last two t e r m s with the values listed in table 1, a n d the fact that this p h o s p h o r is 70% m o r e efficient at 100 kV, so ( n ~ ) = 179.5,
5.2. G e o m e t r i c distortion
T h e two c o m p o u n d lenses u s e d in the relay a r e of e x c e p t i o n a l l y high quality. T h e g e o m e t r i c dist o r t i o n of the system was m e a s u r e d on an optical b e n c h using a sheet of g r a p h p a p e r consisting of 1 m m s q u a r e grids as the object. T h e d i s t o r t i o n is less t h a n 0.2% over the w h o l e field of view ( ~ 24 × 24 mm2). N o b e n d i n g of the lines can be det e c t e d even n e a r the e d g e [24]. 5.3. S h a d i n g distortion
T h e d a r k c u r r e n t of t h e C C D is fairly flat, with the e x c e p t i o n of o n e c o l u m n which has a h i g h e r value of 8 A D U t h a n the rest C C D pixels b u t shows no a b n o r m a l gain v a r i a t i o n w h e n t h e r e is a d e q u a t e illumination, e.g., N > 10, a n d t h e r e fore, is not c o n s i d e r e d a d e f e c t by the m a n u f a c -
G.Y. Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
turer. For low-dose work, it is necessary to correct this by subtracting the bias pattern from the image. The vignetting of the lens system introduces some shading distortion (multiplicative), causing the periphery of an image to be darker than the center. There is about 17% variation from corner to center with uniform illumination. Also, the phosphor grains are visible with uniform illumination. These shading distortions do not significantly distract on-line observation, but the final images should be corrected by a simple gain-normalization process, described by many authors, e.g. refs. [7,8]. With a pixel depth of 14 bits, at least 12 bits can be retained after the gain-normalization process. The images in fig. 2 have been corrected for both additive and multi° plicative shading distortions.
5.4. Resolution The resolution of the phosphor layer was measured using the edge test method. To avoid scratching the phosphor coating of the working screen, the m e a s u r e m e n t was performed on a similarly coated test screen (3 m m thick glass disk 34 m m in diameter). In order to obtain a good accuracy in the measurement, a different lens combination was used providing a magnification of 3.4 x with a CCD camera head (Photometrics CH250) of smaller CCD pixel size (6 x 6 ~ m 2, Kodak KAF1400). The full-width-half-maximum ( F W H M ) of the differential of the knife edge, averaged over 100 lines, was found to be 33 p~m _+ 10% at 200 kV. The M T F of the system was estimated from the power spectrum of images of uniform illumination [25], and the result is in agreement with the phosphor resolution measurement. So the total number of effective pixels is less than 1024 x 1024 with the 1:1 imaging configuration. A demagnification factor of 2 is preferable in order to fully utilize the CCD pixels, at the cost of reduced sensitivity.
5.5. Glare in the lens assembly The interior of the cylinder housing of the lens assembly has been coated with non-reflecting black paint to minimize stray light. However,
27
internal reflections and diffuse scattering cannot be completely eliminated in a lens system. Optical bench tests of this system with a 10 p~m pin-hole as the object and a diffuse light source revealed a background below 0.06% and a halo around the pin-hole about 0.4% of the maximum intensity measured within the pin-hole image. Analysis of T E M diffraction patterns (with a 4 /zm objective aperture) acquired using this system indicated a lower value for the background of 0.03% and a halo of 0.2%. Presumably the lower values obtained in the T E M test are due to the fact that the phosphor screen is a self-illuminating object. Therefore, the final image does not suffer from interference of the blurred image of the light source which would otherwise be formed by the pin-hole. These values indicate that reflections and diffuse scattering in the lens system is not a serious problem for most T E M imaging and diffraction applications.
6. Discussion
Lens coupling has several advantages over the fiber-optic coupling based on authors' experience with both types of coupling schemes. No X-ray irradiation: In a fiber-optically coupled system, X-ray quanta generated in the microscope as well as those generated by electrons striking the scintillator go through the fiber-optic plate and strike the CCD chip, creating bright spots on the image. A group of typically 3 x 3 pixels or less can be affected by one X-ray quantum. The intensity of these spots varies greatly, ranging from just above the dark noise level to saturation of the CCD pixels. The number of these bright spots in an image depends on the high voltage used and integration time. These artifacts can be fixed to some degree by post image-processing, e.g., by replacing the group of pixels with the mean of its surrounding pixels. Another solution would be to record the same image 3 times and take the median of each corresponding pixel to form a new image. Obviously this is not dose efficient and requires that specimen drift is small. The use of longer and leaded fiber-optic rods should lessen the problem, but at
28
G.Y. Fan, M.H. Ellisman /Lens-coupled slow-scan CCD camera
the cost of reduced light transmittance. In contrast, this problem is eliminated in our camera system by "bending" the optical path 90 ° and placing a lead-baffle plate above the camera head, No interference with the microscope t,acuum system: Since the microscope vacuum is sealed by a leaded glass window in our camera and the rest of the system is outside of the microscope, the camera operation as well as reconfiguration do not interfere the microscope vacuum. This is an improvement over most fiber-optically coupled systems where the entire CCD-fiber-optic coupling unit is cooled and is inside the microscope vacuum. The unit acts as a cold trap in the portion of the column maintained with the poorest vacuum and thus requires periodic cleaning. In addition, the units must be protected from condensation and ice formation which would result from venting of the camera chamber area of the microscope. No honey-comb patterns: The honey-comb pattern in a f!ber-optically coupled system, although correctable by the gain-normalization process, interferes with on-line observation where computational speed may not permit on-line gain normalization. The lens-coupled system is free from this type of pattern. Easy to modify or expand." The modular design and flexibility of the lens coupling allows the system to be reconfigured easily. Custom modification or expansion of a fiber-optically coupled system is not so convenient because of its rigid coupling to the fragile and expensive CCD chip. While lens coupling provides the above-mentioned advantages, it suffers two drawbacks when compared with the fiber-optic coupling. The system requires more space and is lower in overall collection efficiency. Further improvement of the collection efficiency by using larger lenses will result in a slightly larger apparatus. The bulkier size does reduce the leg room in the knee space of the microscope but this does not appear to inconvenience microscope operators. 7. Conclusions We have demonstrated that, by choosing proper lens combinations and matching phosphor
scintillator material characteristics to the spectral sensitivity of the CCD imagery it is possible to achieve single-electron detection with a lens-coupled slow-scan CCD camera while still retaining a reasonably large dynamic range. Although the collection efficiency of a few percent of the lens system does not quite match that of a typical fiber-optically coupled CCD system (10%-20%), this does not represent a fundamental limitation. The flexibility and other advantages gained with the lens coupling far outweigh this shortcoming and afford the slow-scan CCD camera a better coupling for T E M applications.
Acknowledgments We thank referee K.-H. H e r r m a n n and Editor P. Kruit for their critical review and comments. We are grateful to Stephen Young and Ronald Milligan for helpful discussions. Assistance from Tektronix, Photometrics and Grant Scientific is gratefully acknowledged. This project was supported by NSF grant DCB 8811713, N I H grants HL27470, NS14718, NS26739, RR04050 and a grant from the H u m a n Frontiers of Science to M.H.E.
References [1] M.E. Mochel and J.M. Mochel, in: Proc. 44th Annual EMSA Meeting, 1986, p. 616. [2] G.Y. Fan and J.H. Butler, in: Computer Simulation of Electron Microscope Diffraction and Images, Eds. W. Krakow and M. O'Keefe (The Minerals, Metals and Materials Society, Warrendale, PA, 1989) p. 195. [3] R.S. Aikens, D.A. Agard and J.W. Sedat, Meth. Cell Biol. 29 (1989) 291. [4] J.C.H. Spence and J.M. Zuo, Rev. Sci. Instr. 59(9)(1988) 2102. [5] P.E. Mooney, G.Y. Fan, C.E. Meyer, K.V. Troung, D.B. Bui and O.L. Krivanek, in: Proc. 12th Int. Congr. for Electron Microscopy, 1990, p. 164. [6] J.F. Mancuso, W.B. Maxwell, R.E. Camp and M.H. Ellisman, in: Proc. 50th Annual EMSA Meeting, Vol. 2, 1992, p. 946. [7] 1. Daberkow, K.-H. Herrmann, L. Liu and W.D. Rau, Ultramicroscopy 38 (1991) 215. [8] S. Kujawa and D. Krahl, Ultramicroscopy 46 (1992) 395. [9] O.L. Krivanek and P.E. Mooney, UItramicroscopy 49 (1992) 95.
G. ]I.. Fan, M.H. Ellisman / L e n s - c o u p l e d slow-scan CCD camera
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