Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1826-1828, May 1975
Scanning Transmission Ion Microscope with a Field Ion Source (ion optics/field ionization/microradiography)
W. H. ESCOVITZ, T. R. FOX, AND R. LEVI-SETTI The Enrico Fermi Institute and Department of Physics, The University of Chicago, Chicago, Illinois 60637
Communicated by Albert V. Crewe, February 24, 1976 ABSTRACT Experiments with a low-resolution scanning transmission ion microscope, using hydrogen ions from a field ionization source, indicate that it will be feasible by this approach to aim at high-resolution ion microscopy. Micrographs of unstained biological specimens have been obtained by critical range absorption of a 55 keV hydrogen ion beam at a resolution of 2000 A.
proached in practice depends essentially on the specific brightness of the ion (or electron) source, since the probe current must exceed that required to obtain an intelligible picture in a practicable scan time. We consider a microscope consisting of a source of radius 6 and an imaging lens of overall magnification M, spherical aberration coefficient C8, and chromatic aberration coefficient Co. We also assume that the source yields ions with energy spread eAV and that its specific brightness j3 is
As a first step toward the development of a high-resolution scanning transmission ion microscope, we have constructed and operated a 65 kV prototype ion-gun scanning transmission ion microscope which accelerates and focuses hydrogen ions from a field ionization (1) source. Our aim is to emulate and complement the achievements of scanning transmission electron microscopy (2), ultimately at comparable resolution, by taking advantage of the interaction properties of fast ions with matter (3). The latter include processes such as charge exchange and molecular ion dissociation which should result in contrast mechanisms unavailable to the electron microscope. We present here experimental data that are relevant to the feasibility of a high-resolution scanning transmission ion microscope and show the first low-resolution images of biological specimens obtained with the prototype instrument. Previous attempts at using ions, and in particular protons, to make images of small objects with conventional electron microscope optics have been reviewed by Grivet (4) and more recently by Levi-Setti (3). The aim was to obtain higher resolution than with electrons. This was believed to be possible because of the ion's shorter De Broglie wavelength. The potential of proton charge exchange as a possible new source of contrast was recognized by Chanson and Magnan (5). Several factors, however, conspired to abort this development of the proton microscope as a high-resolution and viable instrument. In our view, the principal limitations originated in the approach of conventional microscopy itself, where the large rate of proton energy loss (10-30 eV/A in traversing organic materials at 100 keV) causes irreparable chromatic aberration. For this reason, we have chosen the approach of scanning transmission microscopy, where no optics is affected by the beam-specimen interaction so that the latter can be used to advantage in producing contrast. The considerations leading to the optimum parameters and the resulting performance in the scanning transmission electron microscope have been recently reviewed by A. V. Crewe (6) and E. Zeitler (7). We have extended the analysis of these authors to the case of a proton probe (8). The basic problem consists of optimizing the design parameters to obtain the maximum probe current in the smallest beam spot. How closely the theoretical microscope resolution can be ap-
I/r2a262V, [1] where I is the primary current, a the half-angle of emission, and V1 the source voltage. As discussed in ref. 6, the probe current at the focused spot, at overall accelerating voltage V and semi-angle of incidence aj, is (nonrelativistically) ,B =
Jo = 07r2a,262AP2V [2] From [2] we obtain an expression for the radius of the Gaussian image of the source in terms of probe current and specific brightness: rG = 1118 = (Io/3V)i/'(1/irac) [3] with This contribution to the final spot size must be combined those arising from diffraction and from spherical and chromatic aberration. As often done (9), we add these contributions in quadrature and seek the optimum value a0p, of as that reduces the overall probe radius r to its minimum value ropt. It is known that this approach overestimates the final spot size; for our present purposes of establishing the feasibility of a high-resolution scanning transmission ion microscope, however, our result will be in error in a conservative direction. The resulting expressions take a simple form in limiting situations: (a) When the effect of chromatic aberration can be neglected or corrected, we obtain [4] rop = ro8(1 + E2)3/8 with ro,,
=
0.63X 3/4C.51/4
[5]
and
Io 10o [6] 2O3V(0.61X)2 0.30503 where for protons, X = 0.29/V/V (A), and with Io in protons 2
per see and 13 in protons sec'-A-2sr-'V-'. The parameter e here, as in ref. 6, equals the ratio of the radius of the Gaussian
1826
Proc. Nat. Acad. Sci. USA 72
Scanning Transmission Ion Microscope
(1975)
1827
image Mb to that of the Airy disk (to the first zero) from a point source. As can be appreciated from [4] and [6], the theoretical resolution r0, [5 ], can only be approached for very large fl. (b) When the chromatic aberration is dominant in the probe formation, as it is the case in a scanning transmission ion microscope that uses a field ionization ion source (where eAV can amount to several eV) (10), the expression for rpg reduces to rops= roe (1 +
E2)'/'
[7]
where rc
=-
1.1
,AV
xccV
1/2
[8]
We are now in the position to calculate the source brightness needed to reach a certain resolution, if we fix the variables Io, X, C., and C,. In order to obtain a discernible picture with a proton probe, where each proton is contributing a contrast signal, we estimate the need for a minimum probe current of 2.5 X 104 protons per see (500 X 500 point elements, 10 protons per point element, 100 see exposure). Using the values for C, and C, (1.2 and 0.8 cm, respectively) attainable with a superconducting lens of 1.2 cm focal length (3) at 100 kV, we thus estimate, that to reach a probe radius in the high resolution range of 5-10 A, values of # in excess of about 104 protons sec-l-2sr-lV-1 will be needed. Good pictures will require Os in excess of about 10' proton sec-'A-2sr-'V-1. These figures become comparable with those that apply to the scanning transmission electron microscope (6) when multiplied by a factor of order 102 (for thin specimens, this corresponds to the ratio of proton versus electron cross sections for the process originating contrast). The above values of fB are several orders of magnitude larger. than those provided by conventional ion sources. The development of a sufficiently bright ion source is, therefore, the determining factor for the feasibility of a highresolution scanning transmission ion microscope. We have undertaken to experimentally determine whether, by the choice of a field ionization source, this requirement can be met. Our apparatus is schematized in Fig. 1. The optical system is based on the design by Crewe et al. (11) for a field emission electron gun microscope. The specific brightness of the field ion source for our preliminary operating conditions [H2 pressure not greater than 10-4 torr (0.013 Pa), operating limit of the electron multiplier detector] was measured by counting the particles transmitted by the optical system and assuming a virtual source radius of 3.5 A (1). Typical values of # are in the range of 103 protons sec-'A-2sr-'V-1. We have verified that the tip current (and hence fl) increases linearly with the H2 pressure up to at least 2 X 10-' torr (0.26 Pa), so that it should be possible, by differential pumping between the ion source and detector regions, to meet the requirements outlined above for high-resolution work. Field ionization tips, made of (111) oriented W wire, have provided stable microscope operation with lifetimes up to 40 hr. The resolution in the operating conditions of our prototype microscope is limited to 1000-2000 A; this is close to the value calculated from the known energy spread of field-ionized hydrogen (10) and the chromatic aberration coefficient of our simple optical system. Fig. 2 contains our first micrographs of biological specimens taken with hydrogen ions at 55 kV.
DISPLAY TUBE
FIG. 1. Schematic diagram of 63 kV scanning transmission ion gun microscope. The field ionization W tip is cooled by the cryostat to 780K. After evacuation of the microscope to about 10-9 torr (about 1.3 X 10-7 Pa), hydrogen is admitted by a Pd leak to reach an operating pressure of 10-4 torr (0.013 Pa). The tip voltage V1 is in the range 5.5-6.5 kV. The ions are accelerated and focused by a two-electrode Butler lens (11). Two sets of deflecting plates provide for scanning and the correction of astigmatism. A large continuous dynode electron multiplier detects the transmitted beam. The fragmentary appearance of the images shown is due to insufficient statistics per point element. The probe current (103-104 ions/sec), limited by the reduced source operating pressure, is still well below the minimum acceptable level discussed previously. In the transmission mode presently available, high contrast with unstained specimens is achieved mostly by total and partial absorption of the incident beam. In regions of Fig. 2b, c, and d, however, we approach the conditions for microradiography by critical range absorption (3, 12). This obtains when the specimen thickness is within the range straggling distribution of the ion beam for that material. Then the contrast between areas with areal densities above and below the mean range is strongly enhanced. Since both H+ and H2+ ions are present in our beam, there are in fact two range regions where the critical condition occurs. (For soft biological material, the mean range of 55 keV H+ ions is about 0.06 mg/cm2, or 0.6 Mrm, that for the protons from the breakup of 55 keV H2+ ions is about 0.04 mg/cm2 or 0.4 Mm.) Appropriate to thin specimens (20-50 A) and high resolution, several other contrast mechanisms are available to a scanning transmission ion microscope using hydrogen ions. The process of pickup and loss of electrons, by which a sizeable fraction of the beam will emerge after specimen traversal as neutral hydrogen, seems particularly promising for its sensitivity to hydrogen content. The collision dissociation of the H2+ component, upon interaction with the specimen, will provide a unique dark-field signal made out of protons carrying half of the H2+ beam energy. In addition, other processes common to the interaction of ions and electrons with matter will contribute. Details of these, our plans for implementing a high-resolution scanning transmission ion microscope, and
1828
Physics: Escovitz et al.
Proc. Nat. Acad. Sci. USA 72 (1976)
r. .
I . I
I
+
.-
-t
z
.I
C
*
.-,ld"
9c
.'
*.
.,'
.S' .. V.-k 3
FIG. 2. Proton beam micrographs taken at 55 kV, 103-104 ions/sec, 200 lines. (a) Myofibrils from rabbit back muscle. Critical-point dried specimen, unstained, courtesy of M. Lamvik. The individual fibers, about 1 ,um thick, absorb the beam almost completely. Scale bar = 10 Mm. Exposure = 2 min. (b) Nucleus of human lymphocyte, unstained. Critical-point dried specimen, courtesy Dr. H. M. Golomb. Areas of lower mass concentration at the periphery of the nucleus are partially penetrated by the beam. Scale bar = 5 um. Exposure = 1 min. (c) Critical-point dried human chromosomes, unstained. Specimen courtesy of Dr. H. M. Golomb (13). The differential packing of chromatin fibers is detected here in conditions approaching critical range absorption. Scale bar = 5 Mum. Exposure = 2 min. (d) One of the chromosomes in panel c from a higher magnification scan. Scale bar = 1 Mum. Exposure = 4 min.
and a discussion of the limitations from radiation damage have been presented elsewhere (3). Since, however, the fear of fatal radiation damage due to the high rate of proton interaction versus electron interaction seems a common objection to proton microscopy, we wish to summarize our views on this matter. The factor that determines the cumulative damage to any atomic structure in both scanning transmission electron microscope and scanning transmission ion microscope while taking a picture is not the absolute rate of linear energy transfer, but the ratio of useful to damaging signals, e.g., elastic to inelastic scattering. This is of the same order of magnitude for both electrons and protons. Ultimately, the damage is determined by the statistics of contrast-yielding counts per point element, in both instruments. This implies that since proton cross sections are of order 102 larger than for electrons, comparable quality pictures of thin specimens will require scans involving of order 102 fewer protons than electrons and that the radiation damage will be comparable in both cases. In conclusion, we have experimentally established that a field ionization source can provide the brightness required to reach high resolution in a scanning transmission ion microscope. We believe that even at intermediate and low resolution this new instrument will find useful application in the biological, medical, and physical sciences. We thank Prof. A. V. Crewe for his generous assistance in both valuable equipment and advice, and Mr. M. Lamvik and Dr. H. M. Golomb for providing us with the biological specimens. This work is supported by the Louis Block Fund at the University
of Chicago, the Alfred P. Sloan Foundation, the National Science Foundation, and the National Institutes of Health. T.R.F. was a Union Oil Co. of California Foundation Fellow during 19731974. 1. Muller, E. W. & Tsong, T. T. (1969) Field Ion Microscopy (American Elsevier Publ. Co., New York). 2. Crewe, A. V. & Wall, J. (1970) J. Mol. Biol. 48, 375-393. 3. Levi-Setti, R. (1974) "Proton scanning microscopy: Feasibility and promise," in Scanning Electron Microscopy/1974, eds. Johari, 0. & Corvin, I. (lIT Research Institute, Chicago, Ill.), pp. 125-134. 4. Grivet, P. (1972) in Electron Optics (Pergamon Press, Oxford), 2nd English ed., pp. 557-559. 5. Chanson, P. & Magnan, C. (1954) C. R. H. Acad. Sci. 238, 1797-1799; also (1954) Proc. Int. Conf. Electron Microscopy, London, pp. 294-299. 6. Crewe, A. V. (1974) J. Microsc. (Oxford) 100, 247-260. 7. Zeitler, E. (1975) "Scanning transmission electron microscopy," Lectures presented at Centre for Scientific Culture, Erice, Sicily (1973), in Electron Microscopy in Materials Science, ed. Valdr6, U. (Academic Press, New York), in press. 8. Escovitz, W. H., Fox, T. R. & Levi-Setti, R. (1975) Proc. Third Conf. on Applications of Small Accelerators, North Texas State Univ., October, 1974, EFI Report 74-57, in press. 9. Grivet, P. (1972) in Electron Optics (Pergamon Press, Oxford), 2nd English ed., Sect. 16.2.4. 10. Jason, A. J. (1967) Phys. Rev. 156, 266-285. 11. Crewe, A. V., Isaacson, M. & Johnson, D. (1969) Rev. Sci. Instrum. 40, 241-246. 12. Cookson, J. A. (1974) Naturwissenschaften 61, 184-191. 13. Golomb, H. M., Bahr, G. F. & Borgaonkar, D. S. (1971) Genetics 69, 123-128.
Scanning Transmission Ion Microscope with a Field Ion Source (ion optics/field ionization/microradiography)
W. H. ESCOVITZ, T. R. FOX, AND R. LEVI-SETTI The Enrico Fermi Institute and Department of Physics, The University of Chicago, Chicago, Illinois 60637
Communicated by Albert V. Crewe, February 24, 1976 ABSTRACT Experiments with a low-resolution scanning transmission ion microscope, using hydrogen ions from a field ionization source, indicate that it will be feasible by this approach to aim at high-resolution ion microscopy. Micrographs of unstained biological specimens have been obtained by critical range absorption of a 55 keV hydrogen ion beam at a resolution of 2000 A.
proached in practice depends essentially on the specific brightness of the ion (or electron) source, since the probe current must exceed that required to obtain an intelligible picture in a practicable scan time. We consider a microscope consisting of a source of radius 6 and an imaging lens of overall magnification M, spherical aberration coefficient C8, and chromatic aberration coefficient Co. We also assume that the source yields ions with energy spread eAV and that its specific brightness j3 is
As a first step toward the development of a high-resolution scanning transmission ion microscope, we have constructed and operated a 65 kV prototype ion-gun scanning transmission ion microscope which accelerates and focuses hydrogen ions from a field ionization (1) source. Our aim is to emulate and complement the achievements of scanning transmission electron microscopy (2), ultimately at comparable resolution, by taking advantage of the interaction properties of fast ions with matter (3). The latter include processes such as charge exchange and molecular ion dissociation which should result in contrast mechanisms unavailable to the electron microscope. We present here experimental data that are relevant to the feasibility of a high-resolution scanning transmission ion microscope and show the first low-resolution images of biological specimens obtained with the prototype instrument. Previous attempts at using ions, and in particular protons, to make images of small objects with conventional electron microscope optics have been reviewed by Grivet (4) and more recently by Levi-Setti (3). The aim was to obtain higher resolution than with electrons. This was believed to be possible because of the ion's shorter De Broglie wavelength. The potential of proton charge exchange as a possible new source of contrast was recognized by Chanson and Magnan (5). Several factors, however, conspired to abort this development of the proton microscope as a high-resolution and viable instrument. In our view, the principal limitations originated in the approach of conventional microscopy itself, where the large rate of proton energy loss (10-30 eV/A in traversing organic materials at 100 keV) causes irreparable chromatic aberration. For this reason, we have chosen the approach of scanning transmission microscopy, where no optics is affected by the beam-specimen interaction so that the latter can be used to advantage in producing contrast. The considerations leading to the optimum parameters and the resulting performance in the scanning transmission electron microscope have been recently reviewed by A. V. Crewe (6) and E. Zeitler (7). We have extended the analysis of these authors to the case of a proton probe (8). The basic problem consists of optimizing the design parameters to obtain the maximum probe current in the smallest beam spot. How closely the theoretical microscope resolution can be ap-
I/r2a262V, [1] where I is the primary current, a the half-angle of emission, and V1 the source voltage. As discussed in ref. 6, the probe current at the focused spot, at overall accelerating voltage V and semi-angle of incidence aj, is (nonrelativistically) ,B =
Jo = 07r2a,262AP2V [2] From [2] we obtain an expression for the radius of the Gaussian image of the source in terms of probe current and specific brightness: rG = 1118 = (Io/3V)i/'(1/irac) [3] with This contribution to the final spot size must be combined those arising from diffraction and from spherical and chromatic aberration. As often done (9), we add these contributions in quadrature and seek the optimum value a0p, of as that reduces the overall probe radius r to its minimum value ropt. It is known that this approach overestimates the final spot size; for our present purposes of establishing the feasibility of a high-resolution scanning transmission ion microscope, however, our result will be in error in a conservative direction. The resulting expressions take a simple form in limiting situations: (a) When the effect of chromatic aberration can be neglected or corrected, we obtain [4] rop = ro8(1 + E2)3/8 with ro,,
=
0.63X 3/4C.51/4
[5]
and
Io 10o [6] 2O3V(0.61X)2 0.30503 where for protons, X = 0.29/V/V (A), and with Io in protons 2
per see and 13 in protons sec'-A-2sr-'V-'. The parameter e here, as in ref. 6, equals the ratio of the radius of the Gaussian
1826
Proc. Nat. Acad. Sci. USA 72
Scanning Transmission Ion Microscope
(1975)
1827
image Mb to that of the Airy disk (to the first zero) from a point source. As can be appreciated from [4] and [6], the theoretical resolution r0, [5 ], can only be approached for very large fl. (b) When the chromatic aberration is dominant in the probe formation, as it is the case in a scanning transmission ion microscope that uses a field ionization ion source (where eAV can amount to several eV) (10), the expression for rpg reduces to rops= roe (1 +
E2)'/'
[7]
where rc
=-
1.1
,AV
xccV
1/2
[8]
We are now in the position to calculate the source brightness needed to reach a certain resolution, if we fix the variables Io, X, C., and C,. In order to obtain a discernible picture with a proton probe, where each proton is contributing a contrast signal, we estimate the need for a minimum probe current of 2.5 X 104 protons per see (500 X 500 point elements, 10 protons per point element, 100 see exposure). Using the values for C, and C, (1.2 and 0.8 cm, respectively) attainable with a superconducting lens of 1.2 cm focal length (3) at 100 kV, we thus estimate, that to reach a probe radius in the high resolution range of 5-10 A, values of # in excess of about 104 protons sec-l-2sr-lV-1 will be needed. Good pictures will require Os in excess of about 10' proton sec-'A-2sr-'V-1. These figures become comparable with those that apply to the scanning transmission electron microscope (6) when multiplied by a factor of order 102 (for thin specimens, this corresponds to the ratio of proton versus electron cross sections for the process originating contrast). The above values of fB are several orders of magnitude larger. than those provided by conventional ion sources. The development of a sufficiently bright ion source is, therefore, the determining factor for the feasibility of a highresolution scanning transmission ion microscope. We have undertaken to experimentally determine whether, by the choice of a field ionization source, this requirement can be met. Our apparatus is schematized in Fig. 1. The optical system is based on the design by Crewe et al. (11) for a field emission electron gun microscope. The specific brightness of the field ion source for our preliminary operating conditions [H2 pressure not greater than 10-4 torr (0.013 Pa), operating limit of the electron multiplier detector] was measured by counting the particles transmitted by the optical system and assuming a virtual source radius of 3.5 A (1). Typical values of # are in the range of 103 protons sec-'A-2sr-'V-1. We have verified that the tip current (and hence fl) increases linearly with the H2 pressure up to at least 2 X 10-' torr (0.26 Pa), so that it should be possible, by differential pumping between the ion source and detector regions, to meet the requirements outlined above for high-resolution work. Field ionization tips, made of (111) oriented W wire, have provided stable microscope operation with lifetimes up to 40 hr. The resolution in the operating conditions of our prototype microscope is limited to 1000-2000 A; this is close to the value calculated from the known energy spread of field-ionized hydrogen (10) and the chromatic aberration coefficient of our simple optical system. Fig. 2 contains our first micrographs of biological specimens taken with hydrogen ions at 55 kV.
DISPLAY TUBE
FIG. 1. Schematic diagram of 63 kV scanning transmission ion gun microscope. The field ionization W tip is cooled by the cryostat to 780K. After evacuation of the microscope to about 10-9 torr (about 1.3 X 10-7 Pa), hydrogen is admitted by a Pd leak to reach an operating pressure of 10-4 torr (0.013 Pa). The tip voltage V1 is in the range 5.5-6.5 kV. The ions are accelerated and focused by a two-electrode Butler lens (11). Two sets of deflecting plates provide for scanning and the correction of astigmatism. A large continuous dynode electron multiplier detects the transmitted beam. The fragmentary appearance of the images shown is due to insufficient statistics per point element. The probe current (103-104 ions/sec), limited by the reduced source operating pressure, is still well below the minimum acceptable level discussed previously. In the transmission mode presently available, high contrast with unstained specimens is achieved mostly by total and partial absorption of the incident beam. In regions of Fig. 2b, c, and d, however, we approach the conditions for microradiography by critical range absorption (3, 12). This obtains when the specimen thickness is within the range straggling distribution of the ion beam for that material. Then the contrast between areas with areal densities above and below the mean range is strongly enhanced. Since both H+ and H2+ ions are present in our beam, there are in fact two range regions where the critical condition occurs. (For soft biological material, the mean range of 55 keV H+ ions is about 0.06 mg/cm2, or 0.6 Mrm, that for the protons from the breakup of 55 keV H2+ ions is about 0.04 mg/cm2 or 0.4 Mm.) Appropriate to thin specimens (20-50 A) and high resolution, several other contrast mechanisms are available to a scanning transmission ion microscope using hydrogen ions. The process of pickup and loss of electrons, by which a sizeable fraction of the beam will emerge after specimen traversal as neutral hydrogen, seems particularly promising for its sensitivity to hydrogen content. The collision dissociation of the H2+ component, upon interaction with the specimen, will provide a unique dark-field signal made out of protons carrying half of the H2+ beam energy. In addition, other processes common to the interaction of ions and electrons with matter will contribute. Details of these, our plans for implementing a high-resolution scanning transmission ion microscope, and
1828
Physics: Escovitz et al.
Proc. Nat. Acad. Sci. USA 72 (1976)
r. .
I . I
I
+
.-
-t
z
.I
C
*
.-,ld"
9c
.'
*.
.,'
.S' .. V.-k 3
FIG. 2. Proton beam micrographs taken at 55 kV, 103-104 ions/sec, 200 lines. (a) Myofibrils from rabbit back muscle. Critical-point dried specimen, unstained, courtesy of M. Lamvik. The individual fibers, about 1 ,um thick, absorb the beam almost completely. Scale bar = 10 Mm. Exposure = 2 min. (b) Nucleus of human lymphocyte, unstained. Critical-point dried specimen, courtesy Dr. H. M. Golomb. Areas of lower mass concentration at the periphery of the nucleus are partially penetrated by the beam. Scale bar = 5 um. Exposure = 1 min. (c) Critical-point dried human chromosomes, unstained. Specimen courtesy of Dr. H. M. Golomb (13). The differential packing of chromatin fibers is detected here in conditions approaching critical range absorption. Scale bar = 5 Mum. Exposure = 2 min. (d) One of the chromosomes in panel c from a higher magnification scan. Scale bar = 1 Mum. Exposure = 4 min.
and a discussion of the limitations from radiation damage have been presented elsewhere (3). Since, however, the fear of fatal radiation damage due to the high rate of proton interaction versus electron interaction seems a common objection to proton microscopy, we wish to summarize our views on this matter. The factor that determines the cumulative damage to any atomic structure in both scanning transmission electron microscope and scanning transmission ion microscope while taking a picture is not the absolute rate of linear energy transfer, but the ratio of useful to damaging signals, e.g., elastic to inelastic scattering. This is of the same order of magnitude for both electrons and protons. Ultimately, the damage is determined by the statistics of contrast-yielding counts per point element, in both instruments. This implies that since proton cross sections are of order 102 larger than for electrons, comparable quality pictures of thin specimens will require scans involving of order 102 fewer protons than electrons and that the radiation damage will be comparable in both cases. In conclusion, we have experimentally established that a field ionization source can provide the brightness required to reach high resolution in a scanning transmission ion microscope. We believe that even at intermediate and low resolution this new instrument will find useful application in the biological, medical, and physical sciences. We thank Prof. A. V. Crewe for his generous assistance in both valuable equipment and advice, and Mr. M. Lamvik and Dr. H. M. Golomb for providing us with the biological specimens. This work is supported by the Louis Block Fund at the University
of Chicago, the Alfred P. Sloan Foundation, the National Science Foundation, and the National Institutes of Health. T.R.F. was a Union Oil Co. of California Foundation Fellow during 19731974. 1. Muller, E. W. & Tsong, T. T. (1969) Field Ion Microscopy (American Elsevier Publ. Co., New York). 2. Crewe, A. V. & Wall, J. (1970) J. Mol. Biol. 48, 375-393. 3. Levi-Setti, R. (1974) "Proton scanning microscopy: Feasibility and promise," in Scanning Electron Microscopy/1974, eds. Johari, 0. & Corvin, I. (lIT Research Institute, Chicago, Ill.), pp. 125-134. 4. Grivet, P. (1972) in Electron Optics (Pergamon Press, Oxford), 2nd English ed., pp. 557-559. 5. Chanson, P. & Magnan, C. (1954) C. R. H. Acad. Sci. 238, 1797-1799; also (1954) Proc. Int. Conf. Electron Microscopy, London, pp. 294-299. 6. Crewe, A. V. (1974) J. Microsc. (Oxford) 100, 247-260. 7. Zeitler, E. (1975) "Scanning transmission electron microscopy," Lectures presented at Centre for Scientific Culture, Erice, Sicily (1973), in Electron Microscopy in Materials Science, ed. Valdr6, U. (Academic Press, New York), in press. 8. Escovitz, W. H., Fox, T. R. & Levi-Setti, R. (1975) Proc. Third Conf. on Applications of Small Accelerators, North Texas State Univ., October, 1974, EFI Report 74-57, in press. 9. Grivet, P. (1972) in Electron Optics (Pergamon Press, Oxford), 2nd English ed., Sect. 16.2.4. 10. Jason, A. J. (1967) Phys. Rev. 156, 266-285. 11. Crewe, A. V., Isaacson, M. & Johnson, D. (1969) Rev. Sci. Instrum. 40, 241-246. 12. Cookson, J. A. (1974) Naturwissenschaften 61, 184-191. 13. Golomb, H. M., Bahr, G. F. & Borgaonkar, D. S. (1971) Genetics 69, 123-128.
