Scanning xray microscope with 75nm resolution

Scanning xray microscope with 75nm resolution H. Rarback, D. Shu, S. C. Feng, H. Ade, J. Kirz, I. McNulty, D. P. Kern, T. H. P. Chang, Y. Vladimirsky, N. Iskander, D. Attwood, K. McQuaid, and S. Rothman Citation: Review of Scientific Instruments 59, 52 (1988); doi: 10.1063/1.1139965 View online: http://dx.doi.org/10.1063/1.1139965 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/59/1?ver=pdfcov Published by the AIP Publishing

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Scanning x-ray microscope with

15~nm

resolution

H. Rarback, D. Shu,a) and S. C. Feng National Synchrotron Light Source. Brookhaven National Laboratory. Upton. New York 11973

H. Ade, J. Kirz, and L McNulty Physics Department, SUNY Stony Brook, New York 11794

D. P. Kern and T. H. P. Chang IBM Research Center, Yorktown Heights, New York 10598

Y. Vladimirsky Center for X-Ray Optics, Lawrence Berkeley Laboratory and IBM Research Center. Yorktown Heights, New York 10598

N. Iskander and D. Attwood Center for X-Ray Optics, Lawrence Berkeley Laboratory, Berkeley. California 94720

K. McQuaid and S. Rothman Center for X-Ray Optics. Lawrence Berkeley Laboratory and School of Medicine and Dentistry, University afCalifornia, San Francisco, California 94132

(Received 26 August 1987; accepted for publication 29 September 1987) A scanning soft x-ray microscope has been built and operated at the National Synchrotron Light Source. It makes use of a mini-undulator as a bright source of 3.2-nm photons. An electron beam fabricated Fresnel zone plate focuses the beam onto the specimen, which is scanned under computer control. The scanning stage can be moved by both piezoelectric transducers and stepping motors, and the location is monitored by a high-speed laser interferometer. X rays transmitted through the specimen are detected using a flow proportional counter. Images of biological specimens and of artificial microstructures have been made with resolution in the 75-100-nm range. Acquisition time for 256 X 256-pixel images is about 5 min.

INTRODUCTION It has long been realized that soft x-ray microscopes could open up the investigation of unstained, wet biological specimens at resolutions approaching 10 nm. I Efforts to build such instruments have been under way for over 40 years,2 but adequate sources and optical elements were not available until recently. The last 10 years have seen rapid advances in x-ray microscopy, as documented in several reviews;l and conference proceedings. 4 Instruments for contact microscopy are operating in several laboratories using a variety of x-ray sources5 : However, most other types of instruments are designed to operate with synchrotron radiation. We have been developing a scanning microscope. This instrument minimizes the radiation dose to the specimen, generates a digital image, and makes it convenient to keep the specimen in an atmospheric environment. The first suggestions for a scanning x-ray microscope go back to the work of Pattee, 6 but the first instrument used for imaging was built by Horowitz and Howell, which used a pinhole to define the scanning probe? It achieved a resolution of 1 pm, and was the first microscope to operate at a synchrotron radiation source. The first generation of our instrument also used l! l-pm 52

Rev. Sci. Instrum. 59 (1), January 1988

pinhole to define the scanning probe, but used soft x rays and demonstrated the ability to image a wet biological specimen. g More recently we used electron beam fabricated Fresnel zone plates to form submicron probes to generate higherresolution images,') Other scanning instruments using zone plates have been under development at BESSY lO and at Daresbury, II while Spiller has been developing a microscope based on normal incidence mirrors. 12 During the past year we built a completely new instrument with resolution improved by a factor of 2, and speed increased by a factor of 60. This instrument uses the miniundulator at the National Synchrotron Light Source (NSLS) as its x-ray source. Its characteristics are described in Sec. 1. The new zone-plate focusing elements form the subject of Sec. II. The scanning instrument is discussed in Sec. III, while the performance of the system is presented in Sec. IV. In Sec. V we give a few examples of the applications ofthe instrument.

I. THE

X~RA Y

SOURCE

In order for an optical element to form a diffractionlimited probe, it must be coherently illuminated. In the absence of a laboratory source of coherent soft x rays, we must

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@ 1988 American Institute of Physics

52


use collimation and monochromatization to achieve the necessary coherence. We can, therefore, only accept a tiny volume in phase space, whose volume is about V'xV'x'V'yVy'

= ()" 2),

I. Parameters of the rnini-undulator.

TABLE

Type of device Number of periods Period length Range of fundamental Electron beam energy Electron beam current Electron beam size (undulator center)

(1)

where x (y) represents spatial extent, x' (y') angular extent, and A is the wave length,13 with an additional bandwidth restriction MIA of about 1/500 imposed by the use of the zone plate. Since the density of photons in phase space is determined by the source, we must use a source with sufficient brightness to provide the coherent fiux required. For x-ray sources based on electron storage rings, the source brightness is determined by the properties of the electron beam and the magnetic structure that generates the radiation. Undulators are relatively weak, periodic magnetic structures. In combination with low emittance storage rings they have emerged as the brightest laboratory sources of soft x rays. 14 The periodicity ofthe structure gives rise to peaks in the x-ray spectrum, further increasing the fraction of the output power that is usable for our imaging applications. We used the ten period mini-undulator, operating on the 2.S-GeV electron storage ring at the National Synchrotron Light Source. 15 Characteristics of the electron beam and of the magnet structure are summarized in Table I. The undulator was operated with field strength to maximize the flux of x rays with 3.2-nm wavelength. The brightness at this wavelength was measured to be 10 15 photons/(s mm 2 mrad 2 0.1 % BW) in agreement with the design caIculations. 16 The beam line that connects the undulator to the microscope is responsible for creating the necessary coherence properties for the radiation to be focused by the zone plate. It must also satisfy radiation and vacuum safety requirements. The first optical element in the beamline is a water-cooled plane mirror, that deflects the beam by 80 mrad. It also eliminates most of the unwanted hard radiation by absorption due to the poor reflectivity at those wavelengths. Spatial coherence is imposed by a 3OO-,um pinhole located 15 m from

SmCo, hybrid, variable gap 10

8.0cm 1.&-6.0 nrn 2.5 GeV 100 mA typical 350,Lm

230,urad 20{tm 6O,urad

the source. This pinhole is fabricated into a heatpipe-cooled copper block, which absorbs most of the power generated by the undulatof. This pinhole also serves as the entrance slit of an 8-m toroidal grating monochromator (TGM), with a resolving power ;>500. The layout is shown schematically in Fig. 1. To maintain the stability of the x-ray beam a beam position feedback system was built, based on the photoemission signal from two pairs of blades that sense the location of the x-ray beam upstream of the pinhole. 17 This signal was fed back to two groups of four magnets in the storage ring to stabilize the electron orbit through the undulator.

II. THE FOCUSiNG ELEMENT: ZONE PLATE DESIGN AND FABRICATION Fresnel zone plates are circular diffraction gratings. Used in first order, they act as thin lenses, with the focal length given approximately by

f=

(diam)2/4n...t,

(2)

where n is the number of zones. Ditfraction$}imited resolution is the same as that of a lens with the same numerical aperture. 18 The fraction of the incident intensity that the

BEAM POSITION MONITORS ~\

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SHIELD wALL

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FRONT END COMPONENTS 53

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X-ray microscope

Rev. Scl.lnstrum., Vol. 59, No.1, January 1988

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FIG. 2. Side view of zone plate with collimator showing placement of central siop and collimator used to eliminate background radiation.

collw,Cltor open

---------- f

---------------1 I

ideal Fresnel zone plate throws into the first-order focus is 1!(n2) or about 10%. To eliminate unwanted orders, our zone plates were fabricated with a central stop, and a collimator was used between the plate and the focus. The geometry is shown in Fig. 2. The effect of the central stop is to narrow the central focused peak by about 20%, and to throw a larger fraction of the focused intensity into the outer lobes of the Airy pattem. 19 It is a consequence of the geometry described by Eq. (2), that the resolution is approximately equal to the narrowest (outermost) zone of the zone plate. To achieve a resolution of 75 nm, it is essential, therefore, to design and fabricate zone plates with finest zones of 70 nm or less. To maintain near-ideal performance, any random placement errors of the zones must be less than one third the zone width over the entire plate. Furthermore, the zone plate must be coherently illuminated by a source which has a bandwidth that is smaller than 1!1.6n, with n being the number of zones. Various methods have been proposed for the fabrication of high-resolution zone plates, 20 but only three have resulted irt useful focusing elements. These are UV holography,21 electron beam lithography,22-24 and contamination writing.25 The zone plates used in this instrument were fbrmed by electron beam lithography with conventional resist USing a novel single-exposure, double-development!double-plating method. 24 An ultrahigh-resolution vector scan Gaussian beam system designed specifically for nanometer pattern writing 23 •26 was used for this work. The system has a high degree of flexibility in selecting beam potentials (up to 80 kV), field sizes, incrementing rates, etc., and is coupled to a mainframe computer for pattern data preparation and processing. A minimum beam diameter of 8 nm has been achieved with a beam current of 10 pA at 25 kV, To generate the circular shapes needed for the zone plates, a special circular pattern generation technique has been developed. In this technique circular ring segments defined in polar coordinates are used as the basic building blocks (i.e., primitive shapes) fot the pattern instead of rectangles and paraHelo54

Rev. SCi.lnstrum., Vcl. 59, No.1, January 1988

grams in the conventional approach. A real-time high-speed polar/Cartesian conversion is performed to allow the beam to be positioned in the corresponding orthogonal grid addresses of the system. This technique proved to be essential for zone-plate fabrication as it reduces the data volume by several orders of magnitude from the conventional method, while significantly improving pattern quality. In order to block unwanted orders, zone plates for scanning microscopes require a thick centra! absorber. This has been achieved by a single-exposure, double-development! double-plating technique as shown schematically in Fig. 3, in which the blocking region is exposed at twice the dose of the zones. First development (-5 s) clears only the highdose areas and the first plating builds the blocking region. A ~

~

lhf.U HtH nJIll

E-beam exposure

First development and RIE

First electroplating

Second development and R1E

Second electroplating Gold pattern

Resist and plating base removal

FIG. 3. SingJe-exposure double-deve!opment/double-plating process for the zone-plate fabrication.

X-ray mIcroscope

54


second development ( ~ 3 min) clears the opaque zones, and the second plating completes the zone plates. A series of zone plates has been successfully fabricated using this technique with the outer zone width ranging from 100 nm (ZP 100), 70 nm (ZP70), to 50 nm (ZP50). All these zone plates were fabricated on 100 nm Si3N4 membranes with gold as the absorbeL The characteristics of the 70- and 50-nm plates are summarized in Table II. Figure 4 shows a SEM picture of a zone plate from the ZP50 series completed in gold. The central stop is 30 J4-m in diameter and 600 nm thick. In the outer zone region, as shown in Fig. 4, the bar to space ratio is 70/30 nm, and the gold is 100 nm thick. Several other elements of the fabrication process were found to be of particular importance and are summarized: ( 1) setup of the electron beam system in a high-resolution scanning transmission mode for focus and astigmatism corrections, (2) calibration of the electron beam deflection using marks fabricated under laser interferometer control to avoid spatial distortions of the zone-plate patterns causing optical aberrations of the zone-plate lens,24,26 (3) monitoring the development status of the sUboptical resolution zoneplate features using a cross-polarized scattering technique in an optical microscope, and (4) use of low-pressure oxygen reactive ion etching for cleaning the bottom of the developed pattern of resist residues and for activation of the plating base. 24 Furthermore, Moire techniques have been used to inspect the zone plates for proper geometry.24

ill. THE SCANNING MICROSCOPE The apparatus is mounted on a massive granite block that is, in turn, supported on pneumatic mounts to reduce vibrations. Coupling to the monochromator is by a long welded bellows. The x-ray beam emerges from the vacuum passing first through a 150-nm-thick aluminum contamination barrier, then through a 120-nm-thick silicon nitride window. The space between these is pumped by a 120-1/s ion pump. The beam is then incident on the zone plate, mounted on a conventional optical stage. Its alignment relative to the collimator is performed using a pair of x/y adjustTABLE

FIG. 4. Gold Fresnel zone plate with 50-nm nominal outermost zone width. (a) Full view, (b) close-up view of outer zones, with a bar-to-space ratio of 70/30nrn.

ment screws, and is based on the radiation pattern observed past the first-order focus. The specimen is mounted on a standard support that is magnetically held to the scanning stage. In the case of small specimens, electron microscope grids are often used as intermediate supports.

It Zone-plate characteristics.

ZP70 5T4/Lm 28.0f.lm 205 156 1.3mm 2.0/Lm 2.7 >370 gold 130nm 500nm 95/45 nrn 5.9% 2.9% 70nm 70nm 95nm

Outer diameter Central stop diameter Total number of Fresnel zones N umher of working zones Focal length @ 3.2 urn Depth offield @ 3.2 nm Max. allowable tilt I I hl required Material of opaque zones Thickness of opaque zones Thickness of apodized area Bar-to-space ratio (outermost zones) Efficiency (calc., including substr.) Efficiency (exp.) Outermost zone width Knife-edge resolution (theor.) Knife-edge resolution (exp.)

55

Rev. Scl.lnstrum., \101. 59, No.1, January 1988

X-ray microscope

ZP50 62.0/Lm

29.6flm 311 240 l.Umm 1.2 ,urn 2.3

> 500 gold lOOnm 60Dnm

70/30nm 5.2% 1.3% SOnm S2nm 75nm

55

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FIG. 5. The Stony Brook/NSLS Scanning X-ray Microscope. (A) Vacuum snout with Si3 N. window at the end, (D) zone-plate collimator assembly, (e) specimen holder, (D) proportional counter, (E) laser interferometer, (F) piezoelectric translater, and (G) alignment telescope.

To take advantage of the high-resolution focusing elements and the high-brightness source, the scanning instrument must have the necessary position resolution and speed. A new stage was built, based on piezoelectric transducers (PI blocks 27 ), and a laser interferometer. l5 Because the x-ray beam is horizontal, the specimen is mounted in the vertical plane. In scanning, the fast direction is horizontal, and the continuous scan is generated by a programmable waveform generator followed by a high-voltage operation amplifier. The SlOW scan (vertical) is done in steps. The laser interferometer measures the motion of the retroreflectors mounted on: the stage both horizontally and vertically. The least count is 30 nm, and the electronics are capable of handling a lOO-kHz rate, for a maximum scan speed of 3 mm/s. The instrument is enclosed in a Lucite box to reduce air currents and eventually to anow the use of helium in the space around the specimen. A photograph of the apparatus is shown in Fig. 5. To maintain the operation of the interferometer in the plane of the specimen, focusing is accomplished by moving the zone plate and collimator relative to the specimen. The x rays transmitted by the specimen are detected by a flow proportional counter operated with an atmospheric pressure mixture of P 10 gas and helium. The entrance window of the proportional counter is another silicon nitride window, but this one is coated with a thin layer of aluminum to provide a controlled electric field distribution in the important region right behind the window, where the x rays are absorbed and converted to electron-ion pairs which are to be detected. The counter has been operated stably at rates in excess of 1 MHz. In routine operation the count rate did not exceed 300 kHz. The entire scanning stage and counterI scaler system is, under the control of a PDP 11/73 microcomputer via CAMAC. The 11/73 is networked to a MicroVAX II. The latter performs data formatting, processing, and image display operations. 56

Rev. Sci. 'nstrum., Vol. 59, No.1, January 1988

The image is a map of specimen absorptivity. It can be displayed on a color monitor if the absorptivity is translated to a false color scale. Alternatively, more quantitative linescan information can be displayed or plotted. IV. PERfORMANCE A. Intensity of the microprobe The coherent flux incident on the zone plate is measured with the zone plate and collimator removed, and replaced by a pinhole of known diameter. The coherent flux incident on the area of the active zones is 10 MHz for lOO-mA circulating electron current in the storage ring. This value is in agreement with the expected output of the undulator, modified by the efficiency of the mirror, the monochromator, and transmission losses through the contamination barrier, vacuum window, 3 mm of air, and entrance window of the proportional counter. The bandwidth was measured to be -1/500. The efficiency of the zone plates was determined by comparing the flux concentrated into the first-order focus to the flux incident on the zone plate, and thus it includes losses in the substrate (30%). The measured efficiencies of the five zone plates tested varied between 0.75% and 2.9%. These figures are less than the predictions for the ideal zone plate, in part due to the geometry: the opaque zones are generally wider than the spaces between them, as can be seen in Fig. 4(b).

80 Resolution The resolution of the microscope reflects the size of the focal spot generated by the zone plate. To study this quantitatively, we performed a series of knife-edge tests, where a sharp edge was scanned in the focal plane, and the observed X-ray microscope

56


350.0

300.0

1--'---'-'- -'---'--'--'---'--'------.-

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rI~fl\ . ~

2500i'

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N'1~

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1500

r

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! FIG. 6. Typical knife-edge scan with ZP50 1152. It shows a resolution of 75 nm, determined by the drop of the intensity from 75%-25%.

': L~~T-~,~~~J 0.8

1.2

14

1.6

1.8

X··-axis,

2

2.2

2.4

2.6

.2.8

3

3.2

microns

width ofthe transition from transparent to opaque was used to estimate the resolution. By averaging over several scans, we eliminated spurious results due to statistical fluctuations and beam noise. Edges are typically not perfectly sharp, and soft x rays can penetrate through the thinnest regions. Under these circumstances, the results of the scans give an upper limit on the resolution. A typical scan is shown in Fig. 6. It used a nickel mesh as the knife edge. Based on a study of such scans we determined that the resolution of ZP70 is better than 95 nrn and the resolution of ZP50 is better than 75 nm. In view of the uncertainty in the nature of the edge quality and undamped high-frequency vibrations. these values are consistent with the zone plates performing at the expected resolution.

C, Other characteristics We tested the vibration sensitivity of the instrument by positioning a knife edge halfway through the focal spot, and recording the count rate as a function of time. Within the accuracy of the present instrument we have seen no effects due to vibration of the microscope at frequencies less than -20Hz. To test the reproducibility of the operation of the system, we imaged the same irregular edge pattern several times. The same pattern was reproduced to the accuracy of the stage (30 nm). The noise level of the proportional counter and the counting electronics was set with a lower discrimination lev-

FIG. 7. Image of the innermost zones of ZP50 taken with ZP70. Contrast in agreement with the calculations of Buckley (Ref. 25).

57

Rev. SCi.instrum., Vol. 59, No.1, January 1988

51

X-ray microscope

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el to be less than 1 Hz. The signal /background characteristics of the imaging process are, therefore, determined by the quality of the zone plate and the accuracy of the collimator placement. For ZP70 we find a signal/background ratio considerably better than 100.

v. RESULTS AND CONCLUSIONS To demonstrate potential uses of the instrument, we examined both artificial microstructures and biological specimens. An example of the former is shown in Fig. 7. It is the image of some inner zones of a zone plate. The line density in the region shown is 140 nm/line. The zone plate used in forming this image has 70-nm outer line width. The zones are clearly resolved, with a visibility that is consistent with the calculations of Buckley. 28 Fixed and air-dried biological material was deposited on Formvar-coated electron microscope grids. Among the specimens examined were acinar cells from the pancreas of the rat. One such example is shown in Fig. 8. The higher density and granularity on one side of the image is likely related to a concentration of zymogen granules the cell produces. The opaque region at the bottom left is the electron microscope grid bar. A special capability of the microscope is the imaging of wet specimens. Since the mean free path for 3.2-nm x rays in water is only about 4/-tm, the specimen must be placed in a thin wet cell to maintain its aqueous environment, while allowing the x rays to penetrate. In this experiment we formed wet cells from two silicon wafers, each having a 200 X 200lim, 80-nm-thick S(\N4 window. A droplet containing the specimen was sandwiched between these two windows. and the wafers were compressed to expel the excess water. The thickness of the specimen itself defined a minimal-thickness water layer. The wafers were held together by suction and

FIG. 9. Zymogen granule imaged in its unaltered (wet, unstained) state. An interpolation scheme between pixels was used. The diameter of the granule is about 1.2 f-tm.

the edges were sealed off with a thin layer of nail polish. The wet cell was filled with a freshly prepared suspension of zymogen granules isolated from the pancreas of the rat. We examined several granules, one of which is shown in Fig. 9. In several cases the image showed evidence of internal structure. We conclude that the microscope successfully demonstrated that it is capable of routine imaging of microstructures and biological specimens, both after fixation and in their fresh, unaltered state. Image acquisition times were in the range of 1-10 min depending on the image size and the dwell time per pixel. Resolution of 75 nm was demonstrated. The imaging work described in this paper was performed during the month of January 1987. It was a limited

FIG. 8. Acinar cell from the pancreas of the rat. The opaque region on the lower left is the electron microscope grid bar. The higher density on one side of the cell is likely related to the concentration of zymogen granules. (Note the "reversed" color scale. )

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Rev. Scl.lnstrum., Vol. 59, No.1, January 1938

X-ray microscope

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test of the instrument, which is being further improved for installation at the 37 period soft x-ray undulator on the NSLS x-ray ring early in 1988. Along with this more powerful source, a more efficient beamline is being constructed, that will further increase the imaging speed. ACKNOWLEDGMENTS Many people contributed to the work reported here. The mini-undulator was proposed by J. Hastings, and implemented with particular dedication by the NSLS staff. The contributions of H. Hsieh, T. Oversluisen, J. Galayda, G. Van Der Laske, and R. Nawrocky require particular mention. We benefited greatly from the help and encouragement of several colleagues, in particular M. Howells, R. Feder, J. Grendell, and D. Sayre. We acknowledge A. Cox from SSRL for providing us with useful software, and Chris Jacobsen for software development. The NSLS is supported by the Department of Energy Division of Materials Science and Chemical Sciences. Work at Stony Brook is supported by National Science Foundation Grants BBS 8618066 and DMB 8410587. The Center for X-Ray Optics is supported by the Department of Energy. Present address: Institute for High Energy Physics, Academia Sinica, Beijing, Peoples ReplIblic of China. Ip. Lamarque, C. R. Acad. Sci. 202, 684 (1936); H. Wolter, paper presented to the Deutsche Physikalische Gesellschaft, Karlsruhe, 20 Sept. 1951. 2p. Kirkpatrick and H. H. Pattee, Jr., in Handbuch der Physik, edited by S. Fliigge, Vol. 30 (Springer, Berlin, 1957); P. Kirkpatrick and A. V. Baez, J. Opt. Soc. Am. 38, 766 (1948); V. E. Cosslett and W. e. Nixon, X-Ray Microscopy (Cambridge Unlv. Press, Cambridge. 1960). 3E. Spiller, in Handbook on Synchrotron Radiation. Vol. 1, edited by E. E. Koch (North-Holland, Amsterdam, 1983); J. Kirz and D. Sayre, in Synchrotron Radiation Research, edited by H. Winick and S. Doniach (Plenum, New York, 1980); G. Schmahl, Nucl. lnstrum. Methods 208, 361 (1983); J. Kirz and H. Rarback, Rev. Sci. lnstrum. 56, 1 (1985). ·X-Ray Microscopy, edited by G. Schmahl and D. RlIdolph (Springer, Berlin, 1984); X-Ray Microscopy-Instrumentation and Biological Applications, edited by P. e. Cheng and G. J. Jan (Springer, Berlin, 1987). 5p. C. Cheng et ai., Nucl. lnstrum. Methods A246, 668 (1986); R. W. Eason et al., Opt. Acta 33, 50 I (1986). "H. H. Pattee, Jr., J. Opt. Soc. Am. 43,61 (1953); For a modern imp[ementation see D. Mouze, J. Cazaux, and X. Thomas, Ultramicroscopy 17, 269 ( 1985). 7p. Horowitz and J. A. Howell, Science 178, 608 (l9i2); P. Horowitz, Ann. N. Y. Acad. Sci. 306, 203 (1978). gH. Rarback, J. Kenney, J. Kirz, and X. S. Xie, in Scanned Image' Microscopy, edited by A. Ash (Academic, London, 1980), p. 449. ~H. Rarback, J. M. Kenney, J. Kirz, M. Howells, P. Chang, P. J. Coane, R. Feder, P. J. Hnuzego, D. Kern, and D. Sayre, in X-Ray Microscopy, edited by G. Schmahl and D. Rudolph (Springer, Berlin, 1984), p. 203; C. Jacobsen et al., Phys. Med. BioI. 32, 431 (1987). lOB. Niemann, in X-Ray Microscopy, edited by G. Schmahl and D. Rudolph (Springer, Berlin, 1984), p. 217; B. Niemann, in Soft X-ray Optics and

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